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Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces

Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 9, Issue of March 2, pp. 6243–6252, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces* Received for publication, August 29, 2000, and in revised form, October 18, 2000 Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M007876200 Jerzy Majka‡§¶, Jolanta Zakrzewska-Czerwin ˜ ska‡, and Walter Messer§i From the ‡Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Weigla 12, 53-114 Wroclaw, Poland and §Max-Planck-Institut fu ¨ r Molekulare Genetik, Ihnestrasse 73, Berlin-Dahlem D-14195, Germany Using a combined PCR-gel retardation assay, the pre- interaction with the DNA is still poorly understood. The con- ferred recognition sequence of the Streptomyces initia- sensus sequence of the E. coli DnaA box differs depending on tor protein DnaA was determined. The protein showed a the method used for its evaluation. The most stringent defini- preference toward DNA containing two Escherichia co- tion for the DnaA box sequence comes from a determination of li-like DnaA boxes in a head-to-head arrangement (con- binding constants: 59-TT(A/T)TNCACA-39 (7). T ,T ,T , and 2 4 79 sensus sequence TTATCCACA, whereas the consensus T were found to be directly involved in DNA-protein interac- sequence of the DnaA boxes found in the Streptomyces tion (8). E. coli DnaA does not dimerize in solution and inter- oriC region is TTGTCCACA). In quantitative band shift acts with a single DnaA box as a monomer, as measured by gel experiments, the kinetics of the Streptomyces DnaA- retardation (7) and by surface plasmon resonance (9). However, DnaA box interaction was characterized. The DnaA pro- binding to DnaA boxes that differ from the stringent consensus tein can form dimers while binding to a single DnaA box; sequence by one or two base pairs requires two such boxes and dimer formation is mediated by the domain III of the an interaction of DnaA proteins bound to them (9). DnaA binds protein, and the dissociation constant of this process ATP and ADP with high affinity. Both forms of DnaA protein, M. Streptomyces initiator pro- was between 35 and 115 n ATP-DnaA and ADP-DnaA, recognize DNA in a similar fash- tein DnaA interacts in a cooperative manner with DNA ion; however, only ATP-DnaA is active in the DNA replication containing multiple binding sites. For the cooperativity effect, which seems to be independent of the distance process. Recently, it has been shown that E. coli ATP-DnaA separating the DnaA boxes, domain I (or I and II) is protein recognizes also a hexamer sequence, the ATP-DnaA box responsible. The cooperativity constant is moderate and 59-AGATCT-39 or close match of it (9). is in the range of 20 –110. Streptomycetes (Gram-positive soil bacteria) differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large (8-megabase pair), linear and GC-rich (about The initiator protein DnaA plays an essential role in the 72%) chromosome (10, 11). Recent discoveries suggest that initiation of bacterial chromosome replication. The interaction replication of the linear chromosome of Streptomyces coelicolor of DnaA protein with its chromosomal origin (oriC) is best A3(2) proceeds bidirectionally from the centrally located oriC understood in Escherichia coli. The E. coli DnaA protein (52 region toward the ends of the chromosome (12). kDa) binds to five nonpalindromic, nonamer sequences, the The key elements of initiation of the Streptomyces chromo- DnaA boxes. Binding of 10 –20 DnaA monomers promotes a some replication, oriC region and DnaA protein, show higher local unwinding of an adjacent AT-rich region. The unwound complexity than those of E. coli. The Streptomyces oriC region region provides an entry site for the DnaB/DnaC helicase com- contains numerous DnaA boxes, which are grouped into two plex followed by other proteins required to form a replication clusters (13, 14). The Streptomyces DnaA protein consists, like fork (1– 4). all other DnaA proteins, of four domains. In contrast to the Apart from its primary function as a replisome organizer, the other bacteria, the Streptomyces DnaA protein is larger (70 –73 DnaA protein acts as a transcription factor that represses or kDa), since it comprises an additional stretch (;230 amino activates several genes or terminates transcription, depending acids) of predominantly acidic or hydrophobic amino acids on the location and arrangement of DnaA boxes (5). within domain II. The residues lower the isoelectric point of the Both functions of the DnaA protein, replisome organizer and entire Streptomyces DnaA protein (pI 5 5.7) (15, 16). As it was transcription factor, are mediated by its interaction with target shown for the E. coli and Bacillus subtilis DnaA proteins, DNA. The DNA binding domain of E. coli DnaA has been domain III and the C-terminal part (domain IV) of the Strep- localized in the 94 C-terminal amino acids. A potential helix- tomyces DnaA protein are responsible for binding of ATP and loop-helix motif has been reported within this part of the pro- DNA, respectively (15, 17). The consensus sequence of the tein (6). However, because x-ray high resolution structure anal- Streptomyces DnaA box in oriC is 59-TTGTCCACA-39, which ysis is not yet available for DnaA proteins, the detailed differs at the third position (A9G) from the E. coli DnaA box (13). In contrast to E. coli, the Streptomyces DnaA protein can * This work was supported by Deutsche Forschungsgemeinschaft form a dimer when binding to a single DnaA box. Recently, it Grants 436 POL 113/82/0 and Me 659/6 –1 and by Polish Committee of has been shown that the domains I and III are independently Scientific Studies Grant 6 P04A 006 15. The costs of publication of this involved in dimerization of the Streptomyces lividans DnaA article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance protein molecules. The interaction of Streptomyces DnaA pro- with 18 U.S.C. Section 1734 solely to indicate this fact. tein with two DnaA boxes is cooperative and accompanied by Supported by Alexander von Humboldt Foundation Fellowship IV- strong DNA bending (16, 18). However, we do not know the POL 1063505 STP. contribution of the different domains of the Streptomyces DnaA To whom correspondence should be addressed. Tel.: 49-30-8413- 1266; Fax: 49-30-8413-1385; E-mail: [email protected]. protein to cooperative binding. This paper is available on line at http://www.jbc.org 6243 This is an Open Access article under the CC BY license. 6244 Streptomyces DnaA TABLE I In this work, we apply a combined PCR-EMSA technique Oligonucleotides used in the gel retardation experiments (19) to elucidate DNA sequence requirements for Streptomyces The black arrow shows the strong Streptomyces DnaA box; the white DnaA protein binding. Using EMSA, we define details of the arrow shows the E. coli DnaA box R1/R4; the grey arrow shows the box2 binding of the Streptomyces wild type DnaA and its truncated found by the in vitro DNA binding sites selection assay; and the crossed forms to DNA. We reveal that the domain I participates in out arrow shows the scrambled DnaA box. cooperative DnaA protein-DNA interactions. Quantitative analysis of gel mobility shifts allowed us to determine binding constants for dimerization and cooperative DNA protein inter- actions. In addition, we have varied the spacing between two DnaA boxes and examined the consequences on dimerization and cooperative binding of DnaA protein to these boxes. EXPERIMENTAL PROCEDURES Proteins and DNA—The His-tagged wild type DnaA proteins of S. lividans and its truncated mutants DnaA(III-IV) comprising the do- main III and the DNA binding domain were purified on a Ni -nitrilo- triacetic acid-agarose column (Qiagen) as described earlier (15). The DNA binding domain of the DnaA protein DnaA(BD) was expressed as a C-terminal glutathione S-transferase fusion and purified using glu- tathione-Sepharose 4B beads (Amersham Pharmacia Biotech) followed by factor X cleavage as described earlier (16). Oligonucleotides were chemically synthesized and purified by high performance liquid chromatography. For the kinetic analysis of DnaA protein binding complementary nucleotides were annealed by heating at 95 °C for 10 min and gradually cooling to room temperature. The oligonucleotides were end-labeled with [g- P]ATP and T4 polynucle- otide kinase and purified from 5% nondenaturing polyacrylamide gels. In Vitro DNA Binding Site Selection—The recognition motif of the DnaA protein was determined using several cycles of amplification and selection, essentially as described previously (19). The library of 58-bp oligonucleotides for the first selection cycle was prepared by PCR using template oligonucleotides: 59-GGCGGATCCTCGACTAGCGN GCTA- CGAGCTGAGCTCGCG-39 and the primer pair 59-GGCGGATCCTCG- ACTAGCG-39 and 59-CGCGAGTCTAGCTCGTAGC-39 (restriction sites BamHI and SacI are in italic type). The amplification reaction was carried out in 100 ml using 1 pmol of template oligonucleotide (1 pmol corresponds to the number of all possible combinations of 20 degener- The free DNA and complexes were separated by electrophoresis on 5% ated bases) and 100 pmol of each primer for 20 cycles, with each cycle native polyacrylamide gels, prerun for 1 h (0.253 TBE, 6 V/cm, 20 °C). consisting of 15 s at 96 °C, 30 s at 60 °C, and 30 s at 72 °C. Double- Following electrophoresis, the radioactive gel was dried and analyzed stranded oligonucleotides were separated on 3% agarose gels, electro- using a PhosphorImager and ImageQuant software (Molecular Dynam- eluted for1htoTBE buffer, and purified by phenol-chloroform extrac- ics, Inc., Sunnyvale, CA). The variation in experimental data was eval- tion and glycogen-ethanol precipitation. The oligonucleotides were 59- uated by repeating each experiment three or four times. Different end-labeled with [g- P]ATP and T4 polynucleotide kinase and then protein titration experiments showed variations up to 15%. Equilibrium incubated with DnaA protein in concentrations of 5 or 50 nM in binding constants were determined using the modified statistical mechanical buffer (20 mM HEPES-KOH, pH 8.0, 5 mM magnesium acetate, 1 mM model (20) supplemented with the protein dimerization module. For the Na EDTA, 4 mM dithiothreitol, 0.2% Triton X-100, 5 mg/ml bovine DNA substrate containing a single binding site A, the concentration of serum albumin, and 100 mM ATP) The reaction was carried out in 20 ml each species in the gel was expressed by the following equations (model for 30 min at room temperature in the presence of competitor, poly(dI- 1), dC), at a concentration of 2.5 mg/ml. The complexes were separated on S 5 1/Z (Eq. 1) 5% nondenaturing polyacrylamide gels in 0.253 TBE. The bands cor- responding to DNA-protein complexes were excised, and the DNA was SL 5 k L/Z (Eq. 2) A 1 eluted into gel elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl )for3hat60 °Cand recovered by phenol-chloroform extrac- 2 2 SL 5 k k L /Z (Eq. 3) 2 A Di 1 tion followed by glycogen-ethanol precipitation. The DNA was then amplified by PCR at conditions described above and subjected to five 2 Z 5 1 1 k L 1 k k L (Eq. 4) 1 A A Di additional selection cycles of binding and amplification. The subsequent cycles differed only in the amount of competitor added to binding where S, SL, and SL represent concentrations of free DNA, monomer, reactions (for cycles 2– 6, we used 5, 7.5, 20, 50, and 100 mg/ml, respec- and dimer respectively; L is protein (ligand) concentration; k is the tively). Selected oligonucleotides after the fourth, fifth, and sixth round microscopic equilibrium affinity constant for binding site A, and k is Di were digested with BamHI and SacI and cloned into a pUC19 vector. the affinity constant describing the dimerization process. The DNA from independent clones was recovered using a Spin Miniprep For the DNA containing binding sites A and B, the concentrations of Kit (Qiagen) and subjected to sequencing using P-59-end-labeled the species presented in the gel are described by the following equations “247” primer (New England Biolabs) and the Thermo Sequenase cycle (model 2), sequencing kit (Amersham Pharmacia Biotech). S 5 1/Z (Eq. 5) EMSA and Determination of Equilibrium Constants—For binding assays, 59- P-end-labeled DNA (Table I; #0.01 nM) was incubated with SL 5 ~k 1 k !L/Z (Eq. 6) A B 2 different amounts of the DnaA proteins (concentration range indicated in the legend to Fig. 2) in the presence of a competitor (poly(dI-dC), 10 SL 5 ~k @k 1 k # 1 k k k !L /Z (Eq. 7) 2 Di A B A B AB 2 ng/ml) at 20 °C for 30 min in binding buffer (20 mM HEPES-KOH, pH 8.0, 5 mM magnesium acetate, 1 mM Na EDTA, 4 mM dithiothreitol, 2 Z 5 1 1 ~k 1 k !L 1 ~k ~k 1 k ! 1 k k k !L (Eq. 8) 2 A B Di A B A B AB 0.2% Triton X-100, 5 mg/ml bovine serum albumin, and 100 mM ATP). where k is the intrinsic affinity constant for the binding site B, and k B AB is the cooperativity constant describing interaction of protein molecules occupying both binding sites. The abbreviations used are: bp, base pair(s); EMSA, electrophoretic Experimental data were simultaneously fitted to the equations using mobility shift assay; PCR, polymerase chain reaction; SPR, surface TM plasmon resonance. Scientist® for Windows software (MicroMath). Streptomyces DnaA 6245 FIG.1. In vitro DNA binding sites selection assay. A, the experimental de- sign of the test. B, DNA substrate and the primer pair used in the assay. N, any nucleotide; boldface letters, restriction sites BamHI and SacI. C, subsequent rounds of the DnaA binding site determi- nation. Lanes 1, 2, and 3 in each cycle correspond to 0, 5, and 50 nM concentra- tion of the wild-type DnaA protein, re- spectively. Frames indicate gel fragments from which DNA used in the next round and/or sequencing was extracted. * and ** signify DNA bands from which selection toward monomer and dimer started. RESULTS a 5% polyacrylamide nondenaturing gel. Only one protein- bound fraction was visible. The DNA recovered from the com- Determination of the Optimal DnaA Protein Binding Site— plex was amplified by PCR with the pair of primers comple- The consensus sequence of the DnaA box identified within the mentary to the defined sequences of the oligonucleotides (Fig. Streptomyces oriC region is 59-TTGTCCACA-39. The Strepto- 1B) and used as a substrate for the renewed binding assay. In myces oriC region contains a higher number of the DnaA boxes the third selection cycle, we observed a second retarded band (19 boxes) than the E. coli oriC region (five boxes). A DNase I with a higher electrophoretic mobility than the previous one. footprint of the oriC region with DnaA showed protection of all boxes. However, when analyzed individually by surface plas- Here, the bands with lower and higher electrophoretic mobility are called a “dimer” (Fig. 1C,** complex) and a “monomer” (Fig. mon resonance (SPR), only some DnaA boxes were specifically recognized by the DnaA protein (14, 15). To characterize in 1C,* complex), respectively. The DNA from both bands was recovered and used independently in the next selection cycles. detail the recognition properties of the Streptomyces DnaA protein, we applied a binding site selection technique based on The alignments of the selected oligonucleotides after the fourth, fifth, and sixth cycle are shown in Tables II and III. the combined EMSA and polymerase chain reactions (Fig. 1A; Ref. 19). The sequences selected from the “dimer” band after four The purified DnaA protein was incubated with a substrate rounds reveal remarkable features: 63 out of the 67 oligonu- pool of double-stranded oligonucleotides, in which a random cleotides analyzed carry two DnaA boxes, one complete 9-bp 20-bp region was flanked by defined 19-bp sequences contain- DnaA box, the “box 1,” and one incomplete DnaA box, the “box ing restriction sites to facilitate subsequent cloning. The pro- 2” (“box” as defined by sequence). The incomplete box 2 consists tein-DNA complexes were separated from the unbound DNA on of4–7bp(39-part of the DnaA box). Both boxes face each other 6246 Streptomyces DnaA TABLE II Selected DNA binding sites for DnaA (“dimer” band) The sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50 – 85%. Nucleotides that are represented at less than 15% were considered as not significant at the indicated position. Box 1 Box 2 Cycle 4 203 tagcgGCAG TTATCCACA TGTGGAT gctac 103 tagcgG TTATCCACA TGTGTAT GCAgctac 33 tagcgGAAGGG TTGTCCACA TGTGT gctac 23 gtagcCTAGG TTATCCACA TGTGTA cgcta 23 tagcgTTCAGAG TTATCCACC TGTGgcta c 23 tagcgGCGATG TTATCCACA TGTGG gctac 23 tagcg TTGTGCACA TGTGTAT GATTgctac tagcgGCAG TTATCCACA TGAGGAT gctac tagcgAGGT TTATCCACA TGTTGAT gctac tagcgGCAG TTATCCACA TGTGGA Ggctac tagcgGGTTGC TTATCCACA TGTGG gctac tagcgTTGCC TTATGCACA TGTGCA gctac tagcgTTCCTG TTATGCACA TGTGT gctac gtagcCTAAG TTATCCACA TGTGT Gcgcta tagcgGTG TTATGCACA C TGTG AGgctac tagcgCTGG TTTTCCACA TGTGGAT gctac tagcgGG TTTTCCACA GG TGTGCAT gctac tagcgGACC TTGTCCACA TGTGTAT gctac tagcgGGGG TTGTCCACA TGTGTA Cgctac tagcgCA CTGTGCACA TGTGCGTA gctac tagcgGCTCGG ATATCCACA TGTGT gctac tagcgTGTGAG TTATCCCCA TGTGG gctac tagcg GTATCCACA A TGTGTAT GCAgctac gtcgcAA TCATACACA TGTGCA CAcgcta tagcgTGTAG TAGTCCACA TGTG AGgctac tagcgTAATG TTGTGCACA GG TGTCgcta c tagcgGTTTCGG TTGTGCACA TGTg ctac tagcgTGA CCGTCCACA TGTGCGT Ggctac gtcgcCA TCGTCCACA A CCACA CACcgcta gtcgcCACA ATATCCACA AGCGTGAcgcta gtcgcCTAG GTATCCACA AGTTGAAcgcta gtcgcCCACAACC TTATCCCCA GCGcgcta gtcgcCGACG ACTTCCACA GCAGAAcgcta A GAT Consensus sequence NNG TT TCCACA TGTG g ctNN g Tgc Cycle 5 373 tagcgGCAG TTATCCACA TGTGGAT gctac 123 tagcgG TTATCCACA TGTGTAT GCAgctac 43 gtagcCTAgG TTATCCACA TGTGTA cgcta tagcgGTAG TTATCCACA TGTGGAT gctac tagcgGCGG TTATCCACA TGTGGAT gctac tagcgG TCATCCACA TGTGTAT GCAgctac gtagcCTAAG TTATCCACA TGTGTA cgcta gtagcCGAG TTATGCACA AGTGGAT cgcta tagcgCTAG TTATCCACA ACCCGAAgctac AG TA Consensus sequence C G TTATCCACA TGTG AT GC C GT ag Cycle 6 293 tagcgGCAG TTATCCACA TGTGGAT gctac 33 tagcgG TTATCCACA TGTGTAT GCAgctac tagcgG TTATCCACA TGTGCAT GCAgctac tagcgGTAG TTATCCACA TGTGGAT gctac tagcgGCAG TTATCCACA GGTGGAT gctac tagcgGCAG TTACCCACA TGTGGAT gctac gtagcCAAC TTATCCACA GGCACGGcgcta Consensus sequence CAG TTATCCACA TGTGGAT GCTAC Streptomyces DnaA 6247 TABLE III Selected DNA binding sites for DnaA (“monomer” band) The sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50 – 85%. Nucleotides that are represented at less than 15% were considered as not significant at the indicated position. Box 1 Box 2 Cycle 4 203 tagcgGCAG TTATCCACA TGTGGAT gctac 23 tagcgG TTATCCACA TGTGTAT GCAgctac tagcgGCAG TTATCCACA TGTGGA Cgctac tagcgGCGATG TTATCCACA TGTGG gctac tagcgGCGATA TTATCCACA TGTGG gctac gtagcCATGG TTATCCACA TGTGTA cgcta tagcgGTAG TTATCCACA AGTGGAT gctac 83 gtagcCAAC TTATCCACA GGCACGGcgcta 23 gtagcCAAG TTATCCACA ACCCGGAcgcta 23 gtagcCCACACAG TTATCCACA GGCcgcta gtagcCCAGGAC TTATCCACA GGTAcgcta tagcgTACGGG TTATCCACA ACTAGgctac gtagcCGAG TTATCCACA GAATGAAcgcta gtagcACAGAG TTATCCACA GGTTAcgcta tagcGTAG TTATCCACA GATTGGCtac C G T TGGATGC TAC Consensus sequence A TTATCCACA G a c G cacGgcG CtA Cycle 5 83 tagcgGCAG TTATCCACA TGTGGAT gctac 23 tagcgG TTATCCACA TGTGTAT GCAgctac tagcgGCAG TTATCCACA TGTGG GTgctac 193 gtcgcCAAC TTATCCACA GGCACGGcgcta 23 tagcGTAG TTATCCACA GGTTGGCtac 23 gtcgcCTAGG TTATCCACA GGCGTAcgcta tagcgTAG TTATCCACA GATTGgctac gtcgcCTAG TTATCCACA AGCCCTAcgcta tagcgTACTG TTATCCACA GGCTGGgctac gtcgcCTAG TTATCCACA GGCCGTAcgcta gtagcCTAG TTATCCACA GGTCGTAcgcta gtagcACAGAG TTATCCACA GGTTAcgcta gtagcCAAG TTATCCACA ACCCGTAcgcta gtagcCAAG TTATCCACA ACCCGGAcgcta gtagcTCCAGG TTATCCACA GCGCTcgcta gtagcCCCCAAGG TTATCCACA GCTcgcta A G g cacggcg cta Consensus sequence A TTATCCACA G c C t tggttgc tac Cycle 6 tagcgGCAG TTATCCACA GGTGGAT gctac 253 gtagcCAAC TTATCCACA GGCACGGcgcta 43 gtagcCCACACAG TTATCCACA GGCcgcta 33 tagcgTAG TTATCCACA GATTGgctac 23 gtagcCAAG TTATCCACA ACCCGGAcgcta 23 gtagcACAGAG TTATCCACA GGTTAcgcta gtagcCAAC TTATCCACA GGCAGGGcgcta gtagcCAAC TTATCCACA GACACGGcgcta tagcgG TTATCCACA GGTGTATGCAgctac gtagcCAAC TTATCCACA GGTACGGcgcta gtagcCTAG TTATCCACA GGTCGTGcgcta gtagcGCAAC TTATCCACA AGTGGAcgcta cgactAGCGAAG TTATCCACA Gggctac cCc Consensus sequence A A TTATCCACA GG A GGCG CTA gtg and are adjacent (except for a few oligonucleotides). 80% of the selection progressively narrowed the spectrum of observed oli- boxes 1 exhibit the E. coli type of DnaA box (T or A at the third gonucleotide sequences; after six rounds the box 1 is exclusively position), and only 20% of them exhibit the Streptomyces type represented by the E. coli DnaA box R1/R4 (59-TTATCCACA- (G at the third position). During the next two rounds, the 39), and the partial box 2 contains in nearly all cases the 6248 Streptomyces DnaA essential T nucleotides at the 79- and 99-positions. constants for binding sites A and B, respectively; and k is the AB More than half of the oligonucleotides selected from the cooperativity constant describing the interaction of protein monomer band after four rounds contained also two boxes. molecules occupying both binding sites. However, the monomer band had only appeared after the third These equations contain parameters k , k , and k that A B AB round of the selection (Fig. 1C), and therefore it still contained occur only in combination and therefore cannot be unambigu- traces of the oligonucleotides from the dimer band that were ously determined. However, if one of the pair k or k is known, A B subsequently amplified. After the next two selection rounds, it is possible to determine the remaining two. One way to only one out of the 44 oligonucleotides contained two boxes. All achieve that goal is carrying out the gel shift experiment using of the boxes 1 from the monomer band exhibit the E. coli type a substrate containing only one binding site. In our analysis, it DnaA box (R1/R4). is the 1s-0 oligonucleotide. Concentration of free DNA or DNA- The data show that under the conditions of the assay, the protein complexes in this system is given by the Langmuir Streptomyces DnaA protein interacts only with DnaA boxes; no isotherm. other consensus sequences (e.g. the newly identified 6-bp ATP- DnaA box (9)) have been found. The results, presented in Ta- SL 5 1/S 5 k L/~1 1 k L! (Eq. 13) A A bles II and III, show that DnaA protein from Streptomyces The interaction of the 1s-0 DNA fragment with increasing possesses a higher affinity toward DnaA boxes from E. coli amounts of the wild type DnaA and DnaA(III-IV) led to two than toward those from its own oriC region. Binding of the complexes (Fig. 2A, a and c), whereas incubation with the DnaA protein as a dimer apparently requires “head-to-head” DnaA(BD) resulted in a single complex even at the highest orientation of the boxes. protein concentration (Fig. 2A, e). Formation of a dimer band by Kinetic Studies with Streptomyces DnaA Protein—Upstream the first two DnaA derivatives could be explained by an unspe- of the promoter region of the Streptomyces dnaA gene are two cific interaction with the scrambled DnaA box or another se- closely spaced DnaA boxes: a strong one (with the preferred quence motif of the 1s-0 oligonucleotide or a protein dimeriza- Streptomyces sequence: 59-TTGTCCACA-39) and a weak one tion process occurring on the DNA with only one-half of the (59-TTGTCCCCA-39) in head-to-head arrangement with 3 bp in dimer bound to DNA. The first possibility can be excluded, between (21). Interaction of the DnaA protein with these boxes because domain IV alone does not form slower migrating com- creates an autoregulatory circuit similar to that known for the plexes on that substrate, and all three proteins do not interact E. coli dnaA gene (21). Recently, we have shown that binding of with DNA in which both DnaA boxes are scrambled (data not the DnaA protein to both DnaA boxes exhibits a cooperative shown). Taking into account the dimerization process, we mod- character (16). Domains I and III independently participate in ified the original equations by adding a constant describing the the dimerization of the DnaA protein molecules (18). To eval- oligomerization reaction. As a result, we obtained two sets of uate in detail the kinetics of binding of the DnaA protein to equations (model 1 and model 2) for the interaction with a DnaA boxes from the dnaA gene promoter region, we applied single (1s-0) and with two DnaA boxes (2s), respectively (see gel mobility shift assays to determine the binding constants for “Experimental Procedures”). cooperativity as well as for dimerization (20). This assay per- The affinity constant for the single DnaA box, k , and the mits the quantitative analysis of the individual protein-DNA A affinity constant describing the dimerization process, k , were complexes. In our gel retardation experiments, we used the Di calculated by analyzing the interaction of the wild-type DnaA wild-type DnaA protein and its truncated forms, the DnaA(III- (or its truncated forms) with the 1s-0 substrate according to IV) lacking the two N-terminal domains (I and II) and the model 1 or its simplified version, the Langmuir isotherm for the DnaA(BD) containing only the DNA binding domain IV. As a DnaA(BD). The values obtained from those experiments al- prerequisite for the kinetic studies, two DNA substrates con- lowed us to determine k and k , the intrinsic affinity con- taining either a single strong DnaA box, 1s-0 (the weak DnaA B AB stant for the second DnaA box in substrate 2s, and the cooper- box was scrambled), or two DnaA boxes, 2s, derived from the ativity constant, respectively (Fig. 2B). promoter region of the dnaA gene were designed in such a way The wild type DnaA protein and its truncated forms exhibit that the DnaA box(es) is flanked on both sides by sequences similar affinity to the single DnaA box, the 1s-0 substrate corresponding to those within the dnaA promoter region (Table (Table IV; K 5 4 –10 nM). The affinity constants calculated for I). To simplify kinetic calculations, the 2s substrate contains the second DnaA box from the 2s substrate (k ) were found to two identical boxes; the weak DnaA box was replaced by the be nearly identical to the k values. Thus, the affinities seem to strong one (Table I). For the analysis, we used a fixed input be independent of flanking sequences of the DnaA box(es). DNA concentration and various protein concentrations, span- The calculated cooperativity constants, k , showed that ning 4 orders of magnitude (see legend to Fig. 2). The concen- AB only the wild type DnaA is able to interact cooperatively with tration of DNA in the reaction mixture was chosen to be at least the two DnaA boxes; the cooperativity effect increases the 5 times lower than the lowest protein concentration used. affinity of the DnaA protein toward the DnaA boxes over 40 According to the statistical mechanical approach (20), the times. The values obtained for DnaA(III-IV) and DnaA(BD) are interaction between the DnaA protein and the DNA substrate very close to 1; therefore, we consider these interactions as composed of two binding sites could be described by the follow- noncooperative. The results demonstrate that domain I or II or ing equations, both of them are responsible for the cooperativity effect. S 5 1/Z (Eq. 9) The dimerization dissociation constant for the wild-type pro- tein (35 nM) is about 3 times lower than for a protein lacking SL 5 ~k 1 k !L/Z (Eq. 10) A B domains I and II, DnaA(III-IV) (114 nM). The Distance Separating DnaA Boxes Does Not Influence the SL 5 k k k L /Z (Eq. 11) 2 A B AB Cooperativity of the DnaA-DnaA Box Interaction—The arrange- ment of the 19 DnaA boxes occurring within the Streptomyces Z 5 1 1 ~k 1 k !L 1 k k k L (Eq. 12) A B A B AB oriC regions is highly conserved. The spacing between DnaA where S, SL, and SL are concentrations of free DNA, mono- boxes varies from 3 to 20 bp or even more (except for two DnaA boxes that are adjacent). To evaluate the influence of the dis- mer, and dimer bands, respectively; L is protein (ligand) con- centration; k and k are the microscopic equilibrium affinity tance that separates binding sites on the cooperativity, we have A B Streptomyces DnaA 6249 FIG.2. Binding of the DnaA protein forms to Streptomyces DnaA boxes. A, gel retardation experiments with three DnaA protein forms and DNA substrates containing one or two Streptomyces DnaA boxes. The binding to oligonucleotides 1s-0 and 2s of the wild-type protein (a and b), DnaA(III-IV) (c and d), and DnaA(BD) (e and f) are shown. In each panel, protein concentrations are rising from left to right. For DnaA wild type (wt)(a and b), lanes 1–18 indicate 0, 0.1, 0.18, 0.32, 0.57, 1.0, 1.8, 3.2... 1000 nM, respectively; for DnaA(III-IV) and DnaA(BD) (c–f), lanes 1–18 indicate 0, 0.18, 0.32, 0.57, 1.0, 1.8, 3.2, 5.7... 1800 nM. B, quantitative analysis of the DnaA wild type-2s interaction. Intensity (concentration) of every band from the gel presented in A (b) was expressed as a ratio of total lane intensities; band intensity was measured for the area (square) containing a given band and the area separating it from the band with higher mobility. Rhombuses indicate concentrations of TM free DNA; squares and triangles indicate the concentrations of monomer and dimer bands, respectively. Using the Scientist® for Windows program and k and k constants found in band shift assays with a single DnaA box (a, c, and e), the points on the plot were simultaneously fitted A Di to functions describing the concentrations of band species (model 2). Dashed lines present best fitted curves for band concentration functions. designed three double-stranded oligonucleotides with two separating both boxes by 10 bp does not affect their positioning strong DnaA boxes separated by various spacings. The first one on the DNA helix, while in the 2s 15 variant, the two DnaA is the previously analyzed 2s substrate that contains 3 bp boxes lie on the opposite face of the helix. The calculated between the two DnaA boxes. The 2s mimics the spacing be- cooperativity constants for the three substrates are presented tween the fifth and sixth DnaA boxes within the Streptomyces in Table V. oriC region. The other two, 2s 15 and 2s 110 (Table I), contain Adding a turn of the helix (2s 110) increased the cooperat- 5 and 10 additional base pairs between the DnaA boxes, re- ivity about 2.5 times. Surprisingly, the addition of 5 bp (a half spectively. In the 2s 110 substrate, the increase of the distance helix turn) caused negligible change of the cooperativity. As it 6250 Streptomyces DnaA TABLE IV TABLE VI Characterization of DNA binding properties of three DnaA Comparison of the binding properties of wild type DnaA protein to the protein forms to DnaA boxes derived from the promoter region of DnaA boxes derived from the promoter region of the dnaA gene and the dnaA gene oligonucleotides obtained in the optimal site selection For every constant determination, S.D. is given (expressed as a per- For every constant determination, S.D. is given (expressed as a per- centage). *, corresponding dissociation constants K (reciprocal values centage). *, corresponding dissociation constants (reciprocal values of the affinity constants). **, this substrate comprised only one DnaA box; of the affinity constants). k does not exist. ***, the intrinsic affinity constants for the binding sites A and B were found to be not identical. White and black arrows indicate E. coli- and Streptomyces-like DnaA boxes, respectively. The gray arrow indicates box 2 found by the in vitro DNA binding site selection assay. TABLE V Comparison of the cooperativity constants for three DNA substrates that differ in the length of the spacer separating DnaA boxes For every constant determination, S.D. is given (expressed as a per- centage). with two identical boxes, the Sel 1s-1/2 oligonucleotide se- lected after the sixth cycle of the binding assay was also ana- lyzed. The results of this analysis and their comparison with the constants obtained for the wild-type DnaA boxes from the promoter region are summarized in Table VI. The affinity of the wild-type DnaA to the single DnaA box of E. coli is approximately 4 times higher than its affinity to the strong Streptomyces DnaA box. It explains why such DnaA recognition sequences had been found in the selection binding assay. As expected, the dimerization constant does not depend on the DNA recognition sequence; its value is nearly identical for both types of DnaA boxes (Table VI). The cooperativity was shown for the “wild-type” substrate, 2s, the interaction of constant for the Sel 2s substrate is approximately 2 times lower the truncated forms of the DnaA protein, DnaA(III-IV) and than the cooperativity for the wild type Streptomyces DnaA DnaA(BD), with the 2s 15 and 2s 110 substrates exhibited a boxes (2s substrate). This may be due to the fact that the noncooperative character (Table V). selected sequences did not have the 3-bp spacer. Streptomyces DnaA Protein Prefers DnaA Boxes of the E. coli DISCUSSION Type—The binding selection assay suggested that the Strepto- myces DnaA protein prefers the DnaA box of E. coli type (59- Despite extensive work on the mechanism of initiation of TTATCCACA-39) over its own DnaA box (59-TTGTCCACA-39). DNA replication in prokaryotic and eukaryotic systems, sev- To evaluate in detail the interaction of the DnaA protein with eral critical aspects of this mechanism and its control still the E. coli type DnaA box, quantitative gel retardation assays remain obscure. One significant gap is a lack of understanding were performed. As before, we used a two DnaA box system of the biological and biochemical roles of multiple initiator that enabled us to evaluate cooperativity in addition to intrin- protein binding sites required by a large group of plasmid sic affinity constants. Thus, two substrates were used for the (interons) and chromosomal replicons (DnaA boxes). The oriC calculations: Sel 1s-0 and Sel 2s, containing the single box 1 (E. region of the Streptomyces linear chromosome consists of 19 coli DnaA box) or two adjacent boxes 1 in head-to-head orien- DnaA boxes that serve as binding sites. Therefore, Streptomy- tation, respectively (Table I). The Sel 1s-0 substrate was used ces provides a good model for studying the interaction of initi- to determine the affinity constant for the single binding box ator protein with multiple cognate binding sites. The initiator and the dimerization affinity constant. These values were then protein DnaA of Streptomyces contains two dimerization do- used for the interaction analysis of the DnaA protein with two mains (I and III) that are separated by a long flexible domain boxes (substrate Sel 2s). Since the wild-type DnaA protein II. According to our preliminary results (16, 22), interaction of formed dimers during the interaction with a single DnaA box, the DnaA protein with two DnaA boxes exhibits cooperativity the same mathematical models (models 1 and 2; see “Experi- (e.g. the DnaA protein specifically binds to DNA fragments mental Procedures”) were applied. In addition to the substrate with two “weak” DnaA boxes, which are not bound as individ- Streptomyces DnaA 6251 TABLE VII protein interactions that result in cooperative DNA binding Dissociation constants for the DnaA protein interactions with different may lead to bending and/or looping of the intervening DNA, types of DnaA box(es) contributing to the formation of higher order structured DNA- DnaA box(es) K protein complexes. According to our previous studies, the for- nM mation of the Streptomyces initial nucleoprotein complex in- volves the sequential binding of the DnaA protein molecules to Single Streptomyces DnaA box (different types) 10–78 b c R1/R4 E. coli DnaA box 3.4 (2.6) 19 DnaA boxes. Subsequent protein-protein interaction leads to Two Streptomyces DnaA boxes (different types) 1.7–3 bending and looping of the Streptomyces oriC region (18). Three Streptomyces DnaA boxes 1.3 d Our recent experiments indicate that the wild-type Strepto- R1/R4 DnaA box; DnaA protein from E. coli 0.9–1.2 myces DnaA protein binds two or more DnaA boxes in a coop- Intrinsic dissociation constant measured in gel retardation assays erative manner (16). E. coli DnaA protein also binds coopera- (10.4 nM for TTGTTCACA) or apparent dissociation constant from SPR tively to two nonamer DnaA boxes and to three hexamer ATP- assay (12 nM for TTGTTCACA, up to 78 nM for weak DnaA boxes). Apparent dissociation constant measured in SPR assay. DnaA boxes located within the dnaA promoter region (9). Here, Intrinsic dissociation constant measured in gel retardation assay. we have shown that the truncated DnaA protein lacking do- Intrinsic dissociation constant measured in gel retardation assays mains I and II is deficient in cooperative binding to two adja- or apparent dissociation constant from SPR assays. cent sites. Thus, domain I (or domains I and II) is responsible for this effect. However, the domain II of the DnaA proteins is ual boxes). Here, we present the recognition properties of the highly variable and does not contain any relevant secondary Streptomyces DnaA protein and the kinetic details of dimeriza- structure elements. Therefore, it is not a plausible candidate tion and cooperative DNA binding of this protein. Finally, we for any functional role. try to answer the questions of why the Streptomyces oriC region The wild-type DnaA protein binds to two DnaA boxes sepa- contains so many DnaA boxes and how it arose. rated by various spacings with a cooperativity parameter rang- The Streptomyces DnaA Protein Prefers the E. coli DnaA Box ing from 23 to over 100. Surprisingly, cooperativity does not over Its Own DnaA Box—Analysis of 57 DnaA boxes from three depend severely on the spacer length separating both binding different Streptomyces oriC regions showed that the preferred sites (Table V). Adding a turn of the helix (2s 110) increased sequence is 59-TTGTCCACA-39 (“strong” DnaA box). However, the cooperativity about 2.5 times, whereas the addition of 5 bp each of the analyzed oriC regions contains only one strong (a half of a helix turn) had a modest effect on the cooperativity. DnaA box, while the sequences of others, the weak DnaA boxes, Removing of 3 bp (Sel 2s) resulted in only 50% reduction of the differ by one or two bases from the preferred sequence. Our cooperativity parameter. However, the Sel 2s oligonucleotide previous results revealed that the Streptomyces initiator pro- consists of the E. coli type DnaA boxes and therefore cannot be tein DnaA interacts specifically only with a few isolated (14) directly compared with other DNA fragments. The moderate boxes, including the strong one from its own oriC region (Table influence of the spacer length on the cooperativity may suggest VII). To evaluate the recognition properties of the Streptomyces that the protein domains involved in the intramolecular reac- DnaA protein, a binding selection assay based on a combined tions are very flexible. Therefore, changing the spacing be- PCR-EMSA technique was applied. Unexpectedly, we found tween adjacent binding sites does not affect the ability of the that the Streptomyces DnaA protein prefers the E. coli DnaA DnaA protein to bind in a cooperative manner. The spacing box instead of its own DnaA box. After four cycles of binding, between DnaA boxes within the oriC region varies from 3 to 20 only a fraction of the selected oligonucleotides contained Strep- bp. Thus, in theory, the DnaA protein could be able to interact tomyces-like DnaA boxes, whereas after subsequent cycles cooperatively with each pair of the adjacent DnaA boxes. How- (fifth and sixth), this type of DnaA box did dot appear at all. ever, in the binding site selection assay, only DnaA boxes that The results are also supported by the gel retardation experi- face each other have been selected. Therefore, we speculate ments (Table VII). The Streptomyces DnaA protein shows about that cooperativity occurs only when the binding sites are ori- 4 times higher affinity for the E. coli DnaA box (K 5 2.6 nM) ented head-to-head (such a box arrangement has been found in than for its own strong DnaA box (K 5 10.4 nM); therefore, the the promoter region of the dnaA gene as well as in the oriC E. coli box was exclusively selected during the PCR-EMSA regions, e.g. the fifth and sixth DnaA boxes). Our DNase I binding assay. The data obtained by gel retardation are con- footprinting experiments (17) and electron microscopy studies sistent with the dissociation constants determined by surface (18) corroborate this hypothesis. DNase I footprinting experi- plasmon resonance; the affinity of the Streptomyces DnaA pro- ments showed that in the oriC region, at the low protein con- tein to the E. coli DnaA box and to the strong Streptomyces centration, DnaA binds first to the fifth and sixth DnaA boxes. DnaA box have been calculated to be K 5 3.4 nM and K 5 12 d d According to the electron microscopy studies, the highest inci- nM, respectively (Table VII) (16, 17). dence of protein binding occurred at the middle of the first The nucleotides immediately adjacent to the selected binding cluster of DnaA boxes, which corresponds to the location of box exhibit low diversity (59-(A/g)(G/C)-box 1-(G/a)-39); there- DnaA boxes 5 and 6. Therefore, we assume that cooperativity fore, the possibility cannot be excluded that these sequences at close distance determines at least the start of DnaA-oriC influence the binding affinity. The naturally occurring DnaA complex formation. Additional long range interactions may be boxes are usually flanked by G from both sides (59- formed subsequently and may be responsible for loop (a/c/g/t)(G/c)t-DnaA-G-39). formation. Cooperativity and Dimerization: Features of the Streptomyces We established the kinetic constants for dimerization of the DnaA Protein—So far, mainly cooperative DNA binding of transcription factors has been studied extensively (23). Coop- DnaA protein. The Streptomyces DnaA is the first chromosomal initiator for which the kinetics of two dimerization domains, I erativity is best understood for l cI repressor. The recently obtained crystal structure of the cI repressor C-terminal do- and III, have been determined. These domains dimerize inde- main provided a comprehensive study of the molecular basis of pendently (18). The dimerization of the DnaA protein does not cooperativity (24). In eukaryotes, activation of genes frequently occur in the absence of DNA. The wild-type DnaA protein requires the cooperative assembly of large protein complexes reveals 3 times higher dimerization capability than the trun- on the DNA. However, cooperative binding may also serve cated DnaA protein containing only one dimerization domain. functions other than regulation of gene expression. Protein- The intermolecular interactions of the DnaA protein are 6252 Streptomyces DnaA 3–20 times weaker than the interactions between DnaA protein course of Streptomyces evolution, the structure and the se- and its DNA target (Table IV). Probably, it facilitates effective quence of oriC region has been changed. interactions of the DnaA molecules with DNA containing mul- The relatively low affinity of Streptomyces DnaA protein for tiple recognition sequences (e.g. within the oriC region) and a single Streptomyces DnaA box seems to be compensated by a further formation of the nucleoprotein complex. For E. coli high number of DnaA boxes that are bound in a cooperative DnaA, the N-terminal domain 1 has been shown to promote manner. In comparison with the lcI repressor, the cooperativ- oligomerization (25). However, as for Streptomyces DnaA, a ity parameter of the wild-type DnaA protein (40 –100) is mod- second interaction face has been postulated in domain 3 or 4 erate; lcI repressor by binding to its three operator sites shows (26). cooperativity in the range of 250 –950 (20). Such high values in Why Does the Streptomyces oriC Region Contain so Many a system with 19 binding sites (Streptomyces oriC) might cause DnaA Boxes?—The GC content of 57 Streptomyces DnaA boxes irreversible binding of DnaA protein to the oriC region and derived from three Streptomyces oriC regions is about 10 and consequently would block subsequent replication steps. 20% lower than the GC content of the oriC region (63%) and the overall GC content of S. lividans DNA (72%), respectively. REFERENCES However, it is still significantly higher than the GC content of 1. Kornberg, A., and Baker, T. A. (1992) DNA Replication, W. H. Freeman and Co., New York the average E. coli DnaA box (;30%). This difference in GC 2. Skarstad, K., and Boye, E. (1994) Biochim. Biophys. Acta 1217, 111–130 content explains the difference in the DnaA box consensus 3. Messer, W., and Weigel, C. (1996) in Escherichia coli and Salmonella: Cellular sequence between Streptomyces (59-TTGTCCACA-39) and E. and Molecular Biology (Neidhardt, F. C., Curtiss, R., III, Ingraham, J., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, coli (59-TTATCCACA-39). It also results in flanking sequences M., and Umbarger, H. E., eds) pp. 1779 –1601, American Society for Micro- of Streptomyces DnaA boxes that are relatively rich in GC. biology, Washington, D. C. 4. Kaguni, J. M. (1997) Mol. Cells 7, 145–157 As shown in Table VII, the dissociation constant (K ) for 5. Messer, W., and Weigel, C. (1997) Mol. Microbiol. 24, 1– 6 specific binding of individual DnaA boxes derived from the S. 6. Roth, A., and Messer, W. (1995) EMBO J. 14, 2106 –2111 lividans oriC region varies between 10 and 78 nM; a few addi- 7. Schaper, S., and Messer, W. (1995) J. Biol. Chem. 270, 17622–17626 8. Speck, C., Weigel, C., and Messer, W. (1997) Nucleic Acids Res. 25, 3242–3247 tional DnaA boxes are not recognized by DnaA if they are 9. Speck, C., Weigel, C., and Messer, W. (1999) EMBO J. 18, 6169 – 6176 analyzed outside the context of oriC (K exceeding 200 nM). 10. Kutzner, H. J. (1981) in The Prokaryotes (Starr, M. P., Stolp, H., Tru ¨ per, H. G., Interestingly, the affinity of the S. lividans DnaA protein for Balows, A., and Schlegel, H. G., eds) pp. 2028 –2090 Springer, Berlin 11. Lin, Y. S., Kieser, H. M., Hopwood, D. A., and Chen, C. W. (1993) Mol. the R1/R4 E. coli DnaA box (K 5 3.4 nM)is ;3 times higher Microbiol. 10, 923–933 than its affinity for the strong Streptomyces DnaA box. The 12. Musialowski, M. S., Flett, F., Scott, G. B., Hobbs, G., Smith, C. P., and Oliver, S. G. (1994) J. Bacteriol. 176, 5123–5125 apparent dissociation constant for binding of the E. coli DnaA 13. Zakrzewska-Czerwin ˜ ska, J., and Schrempf, H. (1992) J. Bacteriol. 174, protein to the DnaA box R1/R4 was calculated to be K 5 1.1 nM 2688 –2693 (7). 14. Jakimowicz, D., Majka, J., Messer, W., Speck, C., Fernandez, M., Martin, M. C., Sanchez, J., Schauwecker, F., Keller, U., Schrempf, H., and Despite the strong difference in GC content between Strep- Zakrzewska-Czerwin ˜ ska, J. (1998) Microbiology 144, 1281–1290 tomyces and other microoganisms, including E. coli, the do- 15. Majka, J., Messer, W., Schrempf, H., and Zakrzewska-Czerwin ˜ ska, J. (1997) J. mains I and III and the binding domain of the Streptomyces Bacteriol. 179, 2426 –2432 16. Majka, J., Jakimowicz, D., Messer, W., Schrempf, H., Lisowski, M., and DnaA are highly conserved. A consequence of the high GC Zakrzewska-Czerwin ˜ ska, J. (1999) Eur. J. Biochem. 260, 325–335 content of Streptomyces structural genes (72–74%) is the very 17. Majka, J. (1997) Characterization of the Streptomyces lividans initiator protein DnaA, Ph.D. thesis, Ludwik Hirszfeld Institute of Immunology and Exper- nonrandom codon usage, with an extreme paucity of codons imental Therapy, Polish Academy of Sciences, Wroclaw, Poland with A or T in the third position (C or G is usually at the third 18. Jakimowicz, D., Majka, J., Konopa, G., Wgrzyn, G., Messer, W., Schrempf, H., codon position). Like E. coli DnaA protein, the binding domain and Zakrzewska-Czerwin ˜ ska, J. (2000) J. Mol. Biol. 298, 351–364 19. Pollock, R., and Treisman, R. (1990) Nucleic Acids Res. 18, 6197– 6204 of Streptomyces DnaA protein contains the same putative DNA 20. Senear, D. F., and Brenowitz, M. (1991) J. Biol. Chem. 266, 13661–13671 binding motive: two amphipathic a-helices with the basic loop 21. Zakrzewska-Czerwin ˜ ska, J., Nardmann, J., and Schrempf, H. (1994) Mol. Gen. in between followed by a third long a-helix (6, 16). Genet. 242, 440 – 447 22. Jakimowicz, D., Majka, J., Lis, B., Konopa, G., Wgrzyn, G., Messer, W., The affinity of the S. lividans protein for DNA fragments Schrempf, H., and Zakrzewska-Czerwin ˜ ska, J. (2000) Mol. Gen. Genet. 262, containing two or three closely spaced DnaA boxes is 6 –10 1093–1102 23. Jones, S., and Thornton, J. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13–20 times higher than its affinity for the single strong DnaA box 24. Bell, C. E., Frescura, P., Hochschild, A., and Lewis, M. (2000) Cell 101, (Table VII) and is comparable with the affinity of the E. coli 801– 811 DnaA protein to the R1/R4 box (0.9 –1.2). The data suggest that 25. Weigel, C., Schmidt, A., Seitz, H., Tuengler, D., Welzeck, M., and Messer, W. (1999) Mol. Microbiol. 34, 53– 66 efficient binding of the Streptomyces DnaA protein to DNA 26. Messer, W., Blaesing, F., Majka, J., Nardmann, J., Schaper, S., Schmidt, A., requires the presence of more than one Streptomyces DnaA box. Seitz, H., Speck, C., Tu ¨ ngler, D., We ˆ grzyn, G., Weigel, C., Welzeck, M., and Therefore, due to the high GC pressure exerted during the Zakrzewska-Czerwin ˜ ska, J. (1999) Biochimie (Paris) 81, 819 – 825 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces

Majka, Jerzy; Zakrzewska-Czerwiñska, Jolanta; Messer, Walter
Journal of Biological Chemistry – Mar 1, 2001
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 9, Issue of March 2, pp. 6243–6252, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces* Received for publication, August 29, 2000, and in revised form, October 18, 2000 Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M007876200 Jerzy Majka‡§¶, Jolanta Zakrzewska-Czerwin ˜ ska‡, and Walter Messer§i From the ‡Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Weigla 12, 53-114 Wroclaw, Poland and §Max-Planck-Institut fu ¨ r Molekulare Genetik, Ihnestrasse 73, Berlin-Dahlem D-14195, Germany Using a combined PCR-gel retardation assay, the pre- interaction with the DNA is still poorly understood. The con- ferred recognition sequence of the Streptomyces initia- sensus sequence of the E. coli DnaA box differs depending on tor protein DnaA was determined. The protein showed a the method used for its evaluation. The most stringent defini- preference toward DNA containing two Escherichia co- tion for the DnaA box sequence comes from a determination of li-like DnaA boxes in a head-to-head arrangement (con- binding constants: 59-TT(A/T)TNCACA-39 (7). T ,T ,T , and 2 4 79 sensus sequence TTATCCACA, whereas the consensus T were found to be directly involved in DNA-protein interac- sequence of the DnaA boxes found in the Streptomyces tion (8). E. coli DnaA does not dimerize in solution and inter- oriC region is TTGTCCACA). In quantitative band shift acts with a single DnaA box as a monomer, as measured by gel experiments, the kinetics of the Streptomyces DnaA- retardation (7) and by surface plasmon resonance (9). However, DnaA box interaction was characterized. The DnaA pro- binding to DnaA boxes that differ from the stringent consensus tein can form dimers while binding to a single DnaA box; sequence by one or two base pairs requires two such boxes and dimer formation is mediated by the domain III of the an interaction of DnaA proteins bound to them (9). DnaA binds protein, and the dissociation constant of this process ATP and ADP with high affinity. Both forms of DnaA protein, M. Streptomyces initiator pro- was between 35 and 115 n ATP-DnaA and ADP-DnaA, recognize DNA in a similar fash- tein DnaA interacts in a cooperative manner with DNA ion; however, only ATP-DnaA is active in the DNA replication containing multiple binding sites. For the cooperativity effect, which seems to be independent of the distance process. Recently, it has been shown that E. coli ATP-DnaA separating the DnaA boxes, domain I (or I and II) is protein recognizes also a hexamer sequence, the ATP-DnaA box responsible. The cooperativity constant is moderate and 59-AGATCT-39 or close match of it (9). is in the range of 20 –110. Streptomycetes (Gram-positive soil bacteria) differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large (8-megabase pair), linear and GC-rich (about The initiator protein DnaA plays an essential role in the 72%) chromosome (10, 11). Recent discoveries suggest that initiation of bacterial chromosome replication. The interaction replication of the linear chromosome of Streptomyces coelicolor of DnaA protein with its chromosomal origin (oriC) is best A3(2) proceeds bidirectionally from the centrally located oriC understood in Escherichia coli. The E. coli DnaA protein (52 region toward the ends of the chromosome (12). kDa) binds to five nonpalindromic, nonamer sequences, the The key elements of initiation of the Streptomyces chromo- DnaA boxes. Binding of 10 –20 DnaA monomers promotes a some replication, oriC region and DnaA protein, show higher local unwinding of an adjacent AT-rich region. The unwound complexity than those of E. coli. The Streptomyces oriC region region provides an entry site for the DnaB/DnaC helicase com- contains numerous DnaA boxes, which are grouped into two plex followed by other proteins required to form a replication clusters (13, 14). The Streptomyces DnaA protein consists, like fork (1– 4). all other DnaA proteins, of four domains. In contrast to the Apart from its primary function as a replisome organizer, the other bacteria, the Streptomyces DnaA protein is larger (70 –73 DnaA protein acts as a transcription factor that represses or kDa), since it comprises an additional stretch (;230 amino activates several genes or terminates transcription, depending acids) of predominantly acidic or hydrophobic amino acids on the location and arrangement of DnaA boxes (5). within domain II. The residues lower the isoelectric point of the Both functions of the DnaA protein, replisome organizer and entire Streptomyces DnaA protein (pI 5 5.7) (15, 16). As it was transcription factor, are mediated by its interaction with target shown for the E. coli and Bacillus subtilis DnaA proteins, DNA. The DNA binding domain of E. coli DnaA has been domain III and the C-terminal part (domain IV) of the Strep- localized in the 94 C-terminal amino acids. A potential helix- tomyces DnaA protein are responsible for binding of ATP and loop-helix motif has been reported within this part of the pro- DNA, respectively (15, 17). The consensus sequence of the tein (6). However, because x-ray high resolution structure anal- Streptomyces DnaA box in oriC is 59-TTGTCCACA-39, which ysis is not yet available for DnaA proteins, the detailed differs at the third position (A9G) from the E. coli DnaA box (13). In contrast to E. coli, the Streptomyces DnaA protein can * This work was supported by Deutsche Forschungsgemeinschaft form a dimer when binding to a single DnaA box. Recently, it Grants 436 POL 113/82/0 and Me 659/6 –1 and by Polish Committee of has been shown that the domains I and III are independently Scientific Studies Grant 6 P04A 006 15. The costs of publication of this involved in dimerization of the Streptomyces lividans DnaA article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance protein molecules. The interaction of Streptomyces DnaA pro- with 18 U.S.C. Section 1734 solely to indicate this fact. tein with two DnaA boxes is cooperative and accompanied by Supported by Alexander von Humboldt Foundation Fellowship IV- strong DNA bending (16, 18). However, we do not know the POL 1063505 STP. contribution of the different domains of the Streptomyces DnaA To whom correspondence should be addressed. Tel.: 49-30-8413- 1266; Fax: 49-30-8413-1385; E-mail: [email protected]. protein to cooperative binding. This paper is available on line at http://www.jbc.org 6243 This is an Open Access article under the CC BY license. 6244 Streptomyces DnaA TABLE I In this work, we apply a combined PCR-EMSA technique Oligonucleotides used in the gel retardation experiments (19) to elucidate DNA sequence requirements for Streptomyces The black arrow shows the strong Streptomyces DnaA box; the white DnaA protein binding. Using EMSA, we define details of the arrow shows the E. coli DnaA box R1/R4; the grey arrow shows the box2 binding of the Streptomyces wild type DnaA and its truncated found by the in vitro DNA binding sites selection assay; and the crossed forms to DNA. We reveal that the domain I participates in out arrow shows the scrambled DnaA box. cooperative DnaA protein-DNA interactions. Quantitative analysis of gel mobility shifts allowed us to determine binding constants for dimerization and cooperative DNA protein inter- actions. In addition, we have varied the spacing between two DnaA boxes and examined the consequences on dimerization and cooperative binding of DnaA protein to these boxes. EXPERIMENTAL PROCEDURES Proteins and DNA—The His-tagged wild type DnaA proteins of S. lividans and its truncated mutants DnaA(III-IV) comprising the do- main III and the DNA binding domain were purified on a Ni -nitrilo- triacetic acid-agarose column (Qiagen) as described earlier (15). The DNA binding domain of the DnaA protein DnaA(BD) was expressed as a C-terminal glutathione S-transferase fusion and purified using glu- tathione-Sepharose 4B beads (Amersham Pharmacia Biotech) followed by factor X cleavage as described earlier (16). Oligonucleotides were chemically synthesized and purified by high performance liquid chromatography. For the kinetic analysis of DnaA protein binding complementary nucleotides were annealed by heating at 95 °C for 10 min and gradually cooling to room temperature. The oligonucleotides were end-labeled with [g- P]ATP and T4 polynucle- otide kinase and purified from 5% nondenaturing polyacrylamide gels. In Vitro DNA Binding Site Selection—The recognition motif of the DnaA protein was determined using several cycles of amplification and selection, essentially as described previously (19). The library of 58-bp oligonucleotides for the first selection cycle was prepared by PCR using template oligonucleotides: 59-GGCGGATCCTCGACTAGCGN GCTA- CGAGCTGAGCTCGCG-39 and the primer pair 59-GGCGGATCCTCG- ACTAGCG-39 and 59-CGCGAGTCTAGCTCGTAGC-39 (restriction sites BamHI and SacI are in italic type). The amplification reaction was carried out in 100 ml using 1 pmol of template oligonucleotide (1 pmol corresponds to the number of all possible combinations of 20 degener- The free DNA and complexes were separated by electrophoresis on 5% ated bases) and 100 pmol of each primer for 20 cycles, with each cycle native polyacrylamide gels, prerun for 1 h (0.253 TBE, 6 V/cm, 20 °C). consisting of 15 s at 96 °C, 30 s at 60 °C, and 30 s at 72 °C. Double- Following electrophoresis, the radioactive gel was dried and analyzed stranded oligonucleotides were separated on 3% agarose gels, electro- using a PhosphorImager and ImageQuant software (Molecular Dynam- eluted for1htoTBE buffer, and purified by phenol-chloroform extrac- ics, Inc., Sunnyvale, CA). The variation in experimental data was eval- tion and glycogen-ethanol precipitation. The oligonucleotides were 59- uated by repeating each experiment three or four times. Different end-labeled with [g- P]ATP and T4 polynucleotide kinase and then protein titration experiments showed variations up to 15%. Equilibrium incubated with DnaA protein in concentrations of 5 or 50 nM in binding constants were determined using the modified statistical mechanical buffer (20 mM HEPES-KOH, pH 8.0, 5 mM magnesium acetate, 1 mM model (20) supplemented with the protein dimerization module. For the Na EDTA, 4 mM dithiothreitol, 0.2% Triton X-100, 5 mg/ml bovine DNA substrate containing a single binding site A, the concentration of serum albumin, and 100 mM ATP) The reaction was carried out in 20 ml each species in the gel was expressed by the following equations (model for 30 min at room temperature in the presence of competitor, poly(dI- 1), dC), at a concentration of 2.5 mg/ml. The complexes were separated on S 5 1/Z (Eq. 1) 5% nondenaturing polyacrylamide gels in 0.253 TBE. The bands cor- responding to DNA-protein complexes were excised, and the DNA was SL 5 k L/Z (Eq. 2) A 1 eluted into gel elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl )for3hat60 °Cand recovered by phenol-chloroform extrac- 2 2 SL 5 k k L /Z (Eq. 3) 2 A Di 1 tion followed by glycogen-ethanol precipitation. The DNA was then amplified by PCR at conditions described above and subjected to five 2 Z 5 1 1 k L 1 k k L (Eq. 4) 1 A A Di additional selection cycles of binding and amplification. The subsequent cycles differed only in the amount of competitor added to binding where S, SL, and SL represent concentrations of free DNA, monomer, reactions (for cycles 2– 6, we used 5, 7.5, 20, 50, and 100 mg/ml, respec- and dimer respectively; L is protein (ligand) concentration; k is the tively). Selected oligonucleotides after the fourth, fifth, and sixth round microscopic equilibrium affinity constant for binding site A, and k is Di were digested with BamHI and SacI and cloned into a pUC19 vector. the affinity constant describing the dimerization process. The DNA from independent clones was recovered using a Spin Miniprep For the DNA containing binding sites A and B, the concentrations of Kit (Qiagen) and subjected to sequencing using P-59-end-labeled the species presented in the gel are described by the following equations “247” primer (New England Biolabs) and the Thermo Sequenase cycle (model 2), sequencing kit (Amersham Pharmacia Biotech). S 5 1/Z (Eq. 5) EMSA and Determination of Equilibrium Constants—For binding assays, 59- P-end-labeled DNA (Table I; #0.01 nM) was incubated with SL 5 ~k 1 k !L/Z (Eq. 6) A B 2 different amounts of the DnaA proteins (concentration range indicated in the legend to Fig. 2) in the presence of a competitor (poly(dI-dC), 10 SL 5 ~k @k 1 k # 1 k k k !L /Z (Eq. 7) 2 Di A B A B AB 2 ng/ml) at 20 °C for 30 min in binding buffer (20 mM HEPES-KOH, pH 8.0, 5 mM magnesium acetate, 1 mM Na EDTA, 4 mM dithiothreitol, 2 Z 5 1 1 ~k 1 k !L 1 ~k ~k 1 k ! 1 k k k !L (Eq. 8) 2 A B Di A B A B AB 0.2% Triton X-100, 5 mg/ml bovine serum albumin, and 100 mM ATP). where k is the intrinsic affinity constant for the binding site B, and k B AB is the cooperativity constant describing interaction of protein molecules occupying both binding sites. The abbreviations used are: bp, base pair(s); EMSA, electrophoretic Experimental data were simultaneously fitted to the equations using mobility shift assay; PCR, polymerase chain reaction; SPR, surface TM plasmon resonance. Scientist® for Windows software (MicroMath). Streptomyces DnaA 6245 FIG.1. In vitro DNA binding sites selection assay. A, the experimental de- sign of the test. B, DNA substrate and the primer pair used in the assay. N, any nucleotide; boldface letters, restriction sites BamHI and SacI. C, subsequent rounds of the DnaA binding site determi- nation. Lanes 1, 2, and 3 in each cycle correspond to 0, 5, and 50 nM concentra- tion of the wild-type DnaA protein, re- spectively. Frames indicate gel fragments from which DNA used in the next round and/or sequencing was extracted. * and ** signify DNA bands from which selection toward monomer and dimer started. RESULTS a 5% polyacrylamide nondenaturing gel. Only one protein- bound fraction was visible. The DNA recovered from the com- Determination of the Optimal DnaA Protein Binding Site— plex was amplified by PCR with the pair of primers comple- The consensus sequence of the DnaA box identified within the mentary to the defined sequences of the oligonucleotides (Fig. Streptomyces oriC region is 59-TTGTCCACA-39. The Strepto- 1B) and used as a substrate for the renewed binding assay. In myces oriC region contains a higher number of the DnaA boxes the third selection cycle, we observed a second retarded band (19 boxes) than the E. coli oriC region (five boxes). A DNase I with a higher electrophoretic mobility than the previous one. footprint of the oriC region with DnaA showed protection of all boxes. However, when analyzed individually by surface plas- Here, the bands with lower and higher electrophoretic mobility are called a “dimer” (Fig. 1C,** complex) and a “monomer” (Fig. mon resonance (SPR), only some DnaA boxes were specifically recognized by the DnaA protein (14, 15). To characterize in 1C,* complex), respectively. The DNA from both bands was recovered and used independently in the next selection cycles. detail the recognition properties of the Streptomyces DnaA protein, we applied a binding site selection technique based on The alignments of the selected oligonucleotides after the fourth, fifth, and sixth cycle are shown in Tables II and III. the combined EMSA and polymerase chain reactions (Fig. 1A; Ref. 19). The sequences selected from the “dimer” band after four The purified DnaA protein was incubated with a substrate rounds reveal remarkable features: 63 out of the 67 oligonu- pool of double-stranded oligonucleotides, in which a random cleotides analyzed carry two DnaA boxes, one complete 9-bp 20-bp region was flanked by defined 19-bp sequences contain- DnaA box, the “box 1,” and one incomplete DnaA box, the “box ing restriction sites to facilitate subsequent cloning. The pro- 2” (“box” as defined by sequence). The incomplete box 2 consists tein-DNA complexes were separated from the unbound DNA on of4–7bp(39-part of the DnaA box). Both boxes face each other 6246 Streptomyces DnaA TABLE II Selected DNA binding sites for DnaA (“dimer” band) The sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50 – 85%. Nucleotides that are represented at less than 15% were considered as not significant at the indicated position. Box 1 Box 2 Cycle 4 203 tagcgGCAG TTATCCACA TGTGGAT gctac 103 tagcgG TTATCCACA TGTGTAT GCAgctac 33 tagcgGAAGGG TTGTCCACA TGTGT gctac 23 gtagcCTAGG TTATCCACA TGTGTA cgcta 23 tagcgTTCAGAG TTATCCACC TGTGgcta c 23 tagcgGCGATG TTATCCACA TGTGG gctac 23 tagcg TTGTGCACA TGTGTAT GATTgctac tagcgGCAG TTATCCACA TGAGGAT gctac tagcgAGGT TTATCCACA TGTTGAT gctac tagcgGCAG TTATCCACA TGTGGA Ggctac tagcgGGTTGC TTATCCACA TGTGG gctac tagcgTTGCC TTATGCACA TGTGCA gctac tagcgTTCCTG TTATGCACA TGTGT gctac gtagcCTAAG TTATCCACA TGTGT Gcgcta tagcgGTG TTATGCACA C TGTG AGgctac tagcgCTGG TTTTCCACA TGTGGAT gctac tagcgGG TTTTCCACA GG TGTGCAT gctac tagcgGACC TTGTCCACA TGTGTAT gctac tagcgGGGG TTGTCCACA TGTGTA Cgctac tagcgCA CTGTGCACA TGTGCGTA gctac tagcgGCTCGG ATATCCACA TGTGT gctac tagcgTGTGAG TTATCCCCA TGTGG gctac tagcg GTATCCACA A TGTGTAT GCAgctac gtcgcAA TCATACACA TGTGCA CAcgcta tagcgTGTAG TAGTCCACA TGTG AGgctac tagcgTAATG TTGTGCACA GG TGTCgcta c tagcgGTTTCGG TTGTGCACA TGTg ctac tagcgTGA CCGTCCACA TGTGCGT Ggctac gtcgcCA TCGTCCACA A CCACA CACcgcta gtcgcCACA ATATCCACA AGCGTGAcgcta gtcgcCTAG GTATCCACA AGTTGAAcgcta gtcgcCCACAACC TTATCCCCA GCGcgcta gtcgcCGACG ACTTCCACA GCAGAAcgcta A GAT Consensus sequence NNG TT TCCACA TGTG g ctNN g Tgc Cycle 5 373 tagcgGCAG TTATCCACA TGTGGAT gctac 123 tagcgG TTATCCACA TGTGTAT GCAgctac 43 gtagcCTAgG TTATCCACA TGTGTA cgcta tagcgGTAG TTATCCACA TGTGGAT gctac tagcgGCGG TTATCCACA TGTGGAT gctac tagcgG TCATCCACA TGTGTAT GCAgctac gtagcCTAAG TTATCCACA TGTGTA cgcta gtagcCGAG TTATGCACA AGTGGAT cgcta tagcgCTAG TTATCCACA ACCCGAAgctac AG TA Consensus sequence C G TTATCCACA TGTG AT GC C GT ag Cycle 6 293 tagcgGCAG TTATCCACA TGTGGAT gctac 33 tagcgG TTATCCACA TGTGTAT GCAgctac tagcgG TTATCCACA TGTGCAT GCAgctac tagcgGTAG TTATCCACA TGTGGAT gctac tagcgGCAG TTATCCACA GGTGGAT gctac tagcgGCAG TTACCCACA TGTGGAT gctac gtagcCAAC TTATCCACA GGCACGGcgcta Consensus sequence CAG TTATCCACA TGTGGAT GCTAC Streptomyces DnaA 6247 TABLE III Selected DNA binding sites for DnaA (“monomer” band) The sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50 – 85%. Nucleotides that are represented at less than 15% were considered as not significant at the indicated position. Box 1 Box 2 Cycle 4 203 tagcgGCAG TTATCCACA TGTGGAT gctac 23 tagcgG TTATCCACA TGTGTAT GCAgctac tagcgGCAG TTATCCACA TGTGGA Cgctac tagcgGCGATG TTATCCACA TGTGG gctac tagcgGCGATA TTATCCACA TGTGG gctac gtagcCATGG TTATCCACA TGTGTA cgcta tagcgGTAG TTATCCACA AGTGGAT gctac 83 gtagcCAAC TTATCCACA GGCACGGcgcta 23 gtagcCAAG TTATCCACA ACCCGGAcgcta 23 gtagcCCACACAG TTATCCACA GGCcgcta gtagcCCAGGAC TTATCCACA GGTAcgcta tagcgTACGGG TTATCCACA ACTAGgctac gtagcCGAG TTATCCACA GAATGAAcgcta gtagcACAGAG TTATCCACA GGTTAcgcta tagcGTAG TTATCCACA GATTGGCtac C G T TGGATGC TAC Consensus sequence A TTATCCACA G a c G cacGgcG CtA Cycle 5 83 tagcgGCAG TTATCCACA TGTGGAT gctac 23 tagcgG TTATCCACA TGTGTAT GCAgctac tagcgGCAG TTATCCACA TGTGG GTgctac 193 gtcgcCAAC TTATCCACA GGCACGGcgcta 23 tagcGTAG TTATCCACA GGTTGGCtac 23 gtcgcCTAGG TTATCCACA GGCGTAcgcta tagcgTAG TTATCCACA GATTGgctac gtcgcCTAG TTATCCACA AGCCCTAcgcta tagcgTACTG TTATCCACA GGCTGGgctac gtcgcCTAG TTATCCACA GGCCGTAcgcta gtagcCTAG TTATCCACA GGTCGTAcgcta gtagcACAGAG TTATCCACA GGTTAcgcta gtagcCAAG TTATCCACA ACCCGTAcgcta gtagcCAAG TTATCCACA ACCCGGAcgcta gtagcTCCAGG TTATCCACA GCGCTcgcta gtagcCCCCAAGG TTATCCACA GCTcgcta A G g cacggcg cta Consensus sequence A TTATCCACA G c C t tggttgc tac Cycle 6 tagcgGCAG TTATCCACA GGTGGAT gctac 253 gtagcCAAC TTATCCACA GGCACGGcgcta 43 gtagcCCACACAG TTATCCACA GGCcgcta 33 tagcgTAG TTATCCACA GATTGgctac 23 gtagcCAAG TTATCCACA ACCCGGAcgcta 23 gtagcACAGAG TTATCCACA GGTTAcgcta gtagcCAAC TTATCCACA GGCAGGGcgcta gtagcCAAC TTATCCACA GACACGGcgcta tagcgG TTATCCACA GGTGTATGCAgctac gtagcCAAC TTATCCACA GGTACGGcgcta gtagcCTAG TTATCCACA GGTCGTGcgcta gtagcGCAAC TTATCCACA AGTGGAcgcta cgactAGCGAAG TTATCCACA Gggctac cCc Consensus sequence A A TTATCCACA GG A GGCG CTA gtg and are adjacent (except for a few oligonucleotides). 80% of the selection progressively narrowed the spectrum of observed oli- boxes 1 exhibit the E. coli type of DnaA box (T or A at the third gonucleotide sequences; after six rounds the box 1 is exclusively position), and only 20% of them exhibit the Streptomyces type represented by the E. coli DnaA box R1/R4 (59-TTATCCACA- (G at the third position). During the next two rounds, the 39), and the partial box 2 contains in nearly all cases the 6248 Streptomyces DnaA essential T nucleotides at the 79- and 99-positions. constants for binding sites A and B, respectively; and k is the AB More than half of the oligonucleotides selected from the cooperativity constant describing the interaction of protein monomer band after four rounds contained also two boxes. molecules occupying both binding sites. However, the monomer band had only appeared after the third These equations contain parameters k , k , and k that A B AB round of the selection (Fig. 1C), and therefore it still contained occur only in combination and therefore cannot be unambigu- traces of the oligonucleotides from the dimer band that were ously determined. However, if one of the pair k or k is known, A B subsequently amplified. After the next two selection rounds, it is possible to determine the remaining two. One way to only one out of the 44 oligonucleotides contained two boxes. All achieve that goal is carrying out the gel shift experiment using of the boxes 1 from the monomer band exhibit the E. coli type a substrate containing only one binding site. In our analysis, it DnaA box (R1/R4). is the 1s-0 oligonucleotide. Concentration of free DNA or DNA- The data show that under the conditions of the assay, the protein complexes in this system is given by the Langmuir Streptomyces DnaA protein interacts only with DnaA boxes; no isotherm. other consensus sequences (e.g. the newly identified 6-bp ATP- DnaA box (9)) have been found. The results, presented in Ta- SL 5 1/S 5 k L/~1 1 k L! (Eq. 13) A A bles II and III, show that DnaA protein from Streptomyces The interaction of the 1s-0 DNA fragment with increasing possesses a higher affinity toward DnaA boxes from E. coli amounts of the wild type DnaA and DnaA(III-IV) led to two than toward those from its own oriC region. Binding of the complexes (Fig. 2A, a and c), whereas incubation with the DnaA protein as a dimer apparently requires “head-to-head” DnaA(BD) resulted in a single complex even at the highest orientation of the boxes. protein concentration (Fig. 2A, e). Formation of a dimer band by Kinetic Studies with Streptomyces DnaA Protein—Upstream the first two DnaA derivatives could be explained by an unspe- of the promoter region of the Streptomyces dnaA gene are two cific interaction with the scrambled DnaA box or another se- closely spaced DnaA boxes: a strong one (with the preferred quence motif of the 1s-0 oligonucleotide or a protein dimeriza- Streptomyces sequence: 59-TTGTCCACA-39) and a weak one tion process occurring on the DNA with only one-half of the (59-TTGTCCCCA-39) in head-to-head arrangement with 3 bp in dimer bound to DNA. The first possibility can be excluded, between (21). Interaction of the DnaA protein with these boxes because domain IV alone does not form slower migrating com- creates an autoregulatory circuit similar to that known for the plexes on that substrate, and all three proteins do not interact E. coli dnaA gene (21). Recently, we have shown that binding of with DNA in which both DnaA boxes are scrambled (data not the DnaA protein to both DnaA boxes exhibits a cooperative shown). Taking into account the dimerization process, we mod- character (16). Domains I and III independently participate in ified the original equations by adding a constant describing the the dimerization of the DnaA protein molecules (18). To eval- oligomerization reaction. As a result, we obtained two sets of uate in detail the kinetics of binding of the DnaA protein to equations (model 1 and model 2) for the interaction with a DnaA boxes from the dnaA gene promoter region, we applied single (1s-0) and with two DnaA boxes (2s), respectively (see gel mobility shift assays to determine the binding constants for “Experimental Procedures”). cooperativity as well as for dimerization (20). This assay per- The affinity constant for the single DnaA box, k , and the mits the quantitative analysis of the individual protein-DNA A affinity constant describing the dimerization process, k , were complexes. In our gel retardation experiments, we used the Di calculated by analyzing the interaction of the wild-type DnaA wild-type DnaA protein and its truncated forms, the DnaA(III- (or its truncated forms) with the 1s-0 substrate according to IV) lacking the two N-terminal domains (I and II) and the model 1 or its simplified version, the Langmuir isotherm for the DnaA(BD) containing only the DNA binding domain IV. As a DnaA(BD). The values obtained from those experiments al- prerequisite for the kinetic studies, two DNA substrates con- lowed us to determine k and k , the intrinsic affinity con- taining either a single strong DnaA box, 1s-0 (the weak DnaA B AB stant for the second DnaA box in substrate 2s, and the cooper- box was scrambled), or two DnaA boxes, 2s, derived from the ativity constant, respectively (Fig. 2B). promoter region of the dnaA gene were designed in such a way The wild type DnaA protein and its truncated forms exhibit that the DnaA box(es) is flanked on both sides by sequences similar affinity to the single DnaA box, the 1s-0 substrate corresponding to those within the dnaA promoter region (Table (Table IV; K 5 4 –10 nM). The affinity constants calculated for I). To simplify kinetic calculations, the 2s substrate contains the second DnaA box from the 2s substrate (k ) were found to two identical boxes; the weak DnaA box was replaced by the be nearly identical to the k values. Thus, the affinities seem to strong one (Table I). For the analysis, we used a fixed input be independent of flanking sequences of the DnaA box(es). DNA concentration and various protein concentrations, span- The calculated cooperativity constants, k , showed that ning 4 orders of magnitude (see legend to Fig. 2). The concen- AB only the wild type DnaA is able to interact cooperatively with tration of DNA in the reaction mixture was chosen to be at least the two DnaA boxes; the cooperativity effect increases the 5 times lower than the lowest protein concentration used. affinity of the DnaA protein toward the DnaA boxes over 40 According to the statistical mechanical approach (20), the times. The values obtained for DnaA(III-IV) and DnaA(BD) are interaction between the DnaA protein and the DNA substrate very close to 1; therefore, we consider these interactions as composed of two binding sites could be described by the follow- noncooperative. The results demonstrate that domain I or II or ing equations, both of them are responsible for the cooperativity effect. S 5 1/Z (Eq. 9) The dimerization dissociation constant for the wild-type pro- tein (35 nM) is about 3 times lower than for a protein lacking SL 5 ~k 1 k !L/Z (Eq. 10) A B domains I and II, DnaA(III-IV) (114 nM). The Distance Separating DnaA Boxes Does Not Influence the SL 5 k k k L /Z (Eq. 11) 2 A B AB Cooperativity of the DnaA-DnaA Box Interaction—The arrange- ment of the 19 DnaA boxes occurring within the Streptomyces Z 5 1 1 ~k 1 k !L 1 k k k L (Eq. 12) A B A B AB oriC regions is highly conserved. The spacing between DnaA where S, SL, and SL are concentrations of free DNA, mono- boxes varies from 3 to 20 bp or even more (except for two DnaA boxes that are adjacent). To evaluate the influence of the dis- mer, and dimer bands, respectively; L is protein (ligand) con- centration; k and k are the microscopic equilibrium affinity tance that separates binding sites on the cooperativity, we have A B Streptomyces DnaA 6249 FIG.2. Binding of the DnaA protein forms to Streptomyces DnaA boxes. A, gel retardation experiments with three DnaA protein forms and DNA substrates containing one or two Streptomyces DnaA boxes. The binding to oligonucleotides 1s-0 and 2s of the wild-type protein (a and b), DnaA(III-IV) (c and d), and DnaA(BD) (e and f) are shown. In each panel, protein concentrations are rising from left to right. For DnaA wild type (wt)(a and b), lanes 1–18 indicate 0, 0.1, 0.18, 0.32, 0.57, 1.0, 1.8, 3.2... 1000 nM, respectively; for DnaA(III-IV) and DnaA(BD) (c–f), lanes 1–18 indicate 0, 0.18, 0.32, 0.57, 1.0, 1.8, 3.2, 5.7... 1800 nM. B, quantitative analysis of the DnaA wild type-2s interaction. Intensity (concentration) of every band from the gel presented in A (b) was expressed as a ratio of total lane intensities; band intensity was measured for the area (square) containing a given band and the area separating it from the band with higher mobility. Rhombuses indicate concentrations of TM free DNA; squares and triangles indicate the concentrations of monomer and dimer bands, respectively. Using the Scientist® for Windows program and k and k constants found in band shift assays with a single DnaA box (a, c, and e), the points on the plot were simultaneously fitted A Di to functions describing the concentrations of band species (model 2). Dashed lines present best fitted curves for band concentration functions. designed three double-stranded oligonucleotides with two separating both boxes by 10 bp does not affect their positioning strong DnaA boxes separated by various spacings. The first one on the DNA helix, while in the 2s 15 variant, the two DnaA is the previously analyzed 2s substrate that contains 3 bp boxes lie on the opposite face of the helix. The calculated between the two DnaA boxes. The 2s mimics the spacing be- cooperativity constants for the three substrates are presented tween the fifth and sixth DnaA boxes within the Streptomyces in Table V. oriC region. The other two, 2s 15 and 2s 110 (Table I), contain Adding a turn of the helix (2s 110) increased the cooperat- 5 and 10 additional base pairs between the DnaA boxes, re- ivity about 2.5 times. Surprisingly, the addition of 5 bp (a half spectively. In the 2s 110 substrate, the increase of the distance helix turn) caused negligible change of the cooperativity. As it 6250 Streptomyces DnaA TABLE IV TABLE VI Characterization of DNA binding properties of three DnaA Comparison of the binding properties of wild type DnaA protein to the protein forms to DnaA boxes derived from the promoter region of DnaA boxes derived from the promoter region of the dnaA gene and the dnaA gene oligonucleotides obtained in the optimal site selection For every constant determination, S.D. is given (expressed as a per- For every constant determination, S.D. is given (expressed as a per- centage). *, corresponding dissociation constants K (reciprocal values centage). *, corresponding dissociation constants (reciprocal values of the affinity constants). **, this substrate comprised only one DnaA box; of the affinity constants). k does not exist. ***, the intrinsic affinity constants for the binding sites A and B were found to be not identical. White and black arrows indicate E. coli- and Streptomyces-like DnaA boxes, respectively. The gray arrow indicates box 2 found by the in vitro DNA binding site selection assay. TABLE V Comparison of the cooperativity constants for three DNA substrates that differ in the length of the spacer separating DnaA boxes For every constant determination, S.D. is given (expressed as a per- centage). with two identical boxes, the Sel 1s-1/2 oligonucleotide se- lected after the sixth cycle of the binding assay was also ana- lyzed. The results of this analysis and their comparison with the constants obtained for the wild-type DnaA boxes from the promoter region are summarized in Table VI. The affinity of the wild-type DnaA to the single DnaA box of E. coli is approximately 4 times higher than its affinity to the strong Streptomyces DnaA box. It explains why such DnaA recognition sequences had been found in the selection binding assay. As expected, the dimerization constant does not depend on the DNA recognition sequence; its value is nearly identical for both types of DnaA boxes (Table VI). The cooperativity was shown for the “wild-type” substrate, 2s, the interaction of constant for the Sel 2s substrate is approximately 2 times lower the truncated forms of the DnaA protein, DnaA(III-IV) and than the cooperativity for the wild type Streptomyces DnaA DnaA(BD), with the 2s 15 and 2s 110 substrates exhibited a boxes (2s substrate). This may be due to the fact that the noncooperative character (Table V). selected sequences did not have the 3-bp spacer. Streptomyces DnaA Protein Prefers DnaA Boxes of the E. coli DISCUSSION Type—The binding selection assay suggested that the Strepto- myces DnaA protein prefers the DnaA box of E. coli type (59- Despite extensive work on the mechanism of initiation of TTATCCACA-39) over its own DnaA box (59-TTGTCCACA-39). DNA replication in prokaryotic and eukaryotic systems, sev- To evaluate in detail the interaction of the DnaA protein with eral critical aspects of this mechanism and its control still the E. coli type DnaA box, quantitative gel retardation assays remain obscure. One significant gap is a lack of understanding were performed. As before, we used a two DnaA box system of the biological and biochemical roles of multiple initiator that enabled us to evaluate cooperativity in addition to intrin- protein binding sites required by a large group of plasmid sic affinity constants. Thus, two substrates were used for the (interons) and chromosomal replicons (DnaA boxes). The oriC calculations: Sel 1s-0 and Sel 2s, containing the single box 1 (E. region of the Streptomyces linear chromosome consists of 19 coli DnaA box) or two adjacent boxes 1 in head-to-head orien- DnaA boxes that serve as binding sites. Therefore, Streptomy- tation, respectively (Table I). The Sel 1s-0 substrate was used ces provides a good model for studying the interaction of initi- to determine the affinity constant for the single binding box ator protein with multiple cognate binding sites. The initiator and the dimerization affinity constant. These values were then protein DnaA of Streptomyces contains two dimerization do- used for the interaction analysis of the DnaA protein with two mains (I and III) that are separated by a long flexible domain boxes (substrate Sel 2s). Since the wild-type DnaA protein II. According to our preliminary results (16, 22), interaction of formed dimers during the interaction with a single DnaA box, the DnaA protein with two DnaA boxes exhibits cooperativity the same mathematical models (models 1 and 2; see “Experi- (e.g. the DnaA protein specifically binds to DNA fragments mental Procedures”) were applied. In addition to the substrate with two “weak” DnaA boxes, which are not bound as individ- Streptomyces DnaA 6251 TABLE VII protein interactions that result in cooperative DNA binding Dissociation constants for the DnaA protein interactions with different may lead to bending and/or looping of the intervening DNA, types of DnaA box(es) contributing to the formation of higher order structured DNA- DnaA box(es) K protein complexes. According to our previous studies, the for- nM mation of the Streptomyces initial nucleoprotein complex in- volves the sequential binding of the DnaA protein molecules to Single Streptomyces DnaA box (different types) 10–78 b c R1/R4 E. coli DnaA box 3.4 (2.6) 19 DnaA boxes. Subsequent protein-protein interaction leads to Two Streptomyces DnaA boxes (different types) 1.7–3 bending and looping of the Streptomyces oriC region (18). Three Streptomyces DnaA boxes 1.3 d Our recent experiments indicate that the wild-type Strepto- R1/R4 DnaA box; DnaA protein from E. coli 0.9–1.2 myces DnaA protein binds two or more DnaA boxes in a coop- Intrinsic dissociation constant measured in gel retardation assays erative manner (16). E. coli DnaA protein also binds coopera- (10.4 nM for TTGTTCACA) or apparent dissociation constant from SPR tively to two nonamer DnaA boxes and to three hexamer ATP- assay (12 nM for TTGTTCACA, up to 78 nM for weak DnaA boxes). Apparent dissociation constant measured in SPR assay. DnaA boxes located within the dnaA promoter region (9). Here, Intrinsic dissociation constant measured in gel retardation assay. we have shown that the truncated DnaA protein lacking do- Intrinsic dissociation constant measured in gel retardation assays mains I and II is deficient in cooperative binding to two adja- or apparent dissociation constant from SPR assays. cent sites. Thus, domain I (or domains I and II) is responsible for this effect. However, the domain II of the DnaA proteins is ual boxes). Here, we present the recognition properties of the highly variable and does not contain any relevant secondary Streptomyces DnaA protein and the kinetic details of dimeriza- structure elements. Therefore, it is not a plausible candidate tion and cooperative DNA binding of this protein. Finally, we for any functional role. try to answer the questions of why the Streptomyces oriC region The wild-type DnaA protein binds to two DnaA boxes sepa- contains so many DnaA boxes and how it arose. rated by various spacings with a cooperativity parameter rang- The Streptomyces DnaA Protein Prefers the E. coli DnaA Box ing from 23 to over 100. Surprisingly, cooperativity does not over Its Own DnaA Box—Analysis of 57 DnaA boxes from three depend severely on the spacer length separating both binding different Streptomyces oriC regions showed that the preferred sites (Table V). Adding a turn of the helix (2s 110) increased sequence is 59-TTGTCCACA-39 (“strong” DnaA box). However, the cooperativity about 2.5 times, whereas the addition of 5 bp each of the analyzed oriC regions contains only one strong (a half of a helix turn) had a modest effect on the cooperativity. DnaA box, while the sequences of others, the weak DnaA boxes, Removing of 3 bp (Sel 2s) resulted in only 50% reduction of the differ by one or two bases from the preferred sequence. Our cooperativity parameter. However, the Sel 2s oligonucleotide previous results revealed that the Streptomyces initiator pro- consists of the E. coli type DnaA boxes and therefore cannot be tein DnaA interacts specifically only with a few isolated (14) directly compared with other DNA fragments. The moderate boxes, including the strong one from its own oriC region (Table influence of the spacer length on the cooperativity may suggest VII). To evaluate the recognition properties of the Streptomyces that the protein domains involved in the intramolecular reac- DnaA protein, a binding selection assay based on a combined tions are very flexible. Therefore, changing the spacing be- PCR-EMSA technique was applied. Unexpectedly, we found tween adjacent binding sites does not affect the ability of the that the Streptomyces DnaA protein prefers the E. coli DnaA DnaA protein to bind in a cooperative manner. The spacing box instead of its own DnaA box. After four cycles of binding, between DnaA boxes within the oriC region varies from 3 to 20 only a fraction of the selected oligonucleotides contained Strep- bp. Thus, in theory, the DnaA protein could be able to interact tomyces-like DnaA boxes, whereas after subsequent cycles cooperatively with each pair of the adjacent DnaA boxes. How- (fifth and sixth), this type of DnaA box did dot appear at all. ever, in the binding site selection assay, only DnaA boxes that The results are also supported by the gel retardation experi- face each other have been selected. Therefore, we speculate ments (Table VII). The Streptomyces DnaA protein shows about that cooperativity occurs only when the binding sites are ori- 4 times higher affinity for the E. coli DnaA box (K 5 2.6 nM) ented head-to-head (such a box arrangement has been found in than for its own strong DnaA box (K 5 10.4 nM); therefore, the the promoter region of the dnaA gene as well as in the oriC E. coli box was exclusively selected during the PCR-EMSA regions, e.g. the fifth and sixth DnaA boxes). Our DNase I binding assay. The data obtained by gel retardation are con- footprinting experiments (17) and electron microscopy studies sistent with the dissociation constants determined by surface (18) corroborate this hypothesis. DNase I footprinting experi- plasmon resonance; the affinity of the Streptomyces DnaA pro- ments showed that in the oriC region, at the low protein con- tein to the E. coli DnaA box and to the strong Streptomyces centration, DnaA binds first to the fifth and sixth DnaA boxes. DnaA box have been calculated to be K 5 3.4 nM and K 5 12 d d According to the electron microscopy studies, the highest inci- nM, respectively (Table VII) (16, 17). dence of protein binding occurred at the middle of the first The nucleotides immediately adjacent to the selected binding cluster of DnaA boxes, which corresponds to the location of box exhibit low diversity (59-(A/g)(G/C)-box 1-(G/a)-39); there- DnaA boxes 5 and 6. Therefore, we assume that cooperativity fore, the possibility cannot be excluded that these sequences at close distance determines at least the start of DnaA-oriC influence the binding affinity. The naturally occurring DnaA complex formation. Additional long range interactions may be boxes are usually flanked by G from both sides (59- formed subsequently and may be responsible for loop (a/c/g/t)(G/c)t-DnaA-G-39). formation. Cooperativity and Dimerization: Features of the Streptomyces We established the kinetic constants for dimerization of the DnaA Protein—So far, mainly cooperative DNA binding of transcription factors has been studied extensively (23). Coop- DnaA protein. The Streptomyces DnaA is the first chromosomal initiator for which the kinetics of two dimerization domains, I erativity is best understood for l cI repressor. The recently obtained crystal structure of the cI repressor C-terminal do- and III, have been determined. These domains dimerize inde- main provided a comprehensive study of the molecular basis of pendently (18). The dimerization of the DnaA protein does not cooperativity (24). In eukaryotes, activation of genes frequently occur in the absence of DNA. The wild-type DnaA protein requires the cooperative assembly of large protein complexes reveals 3 times higher dimerization capability than the trun- on the DNA. However, cooperative binding may also serve cated DnaA protein containing only one dimerization domain. functions other than regulation of gene expression. Protein- The intermolecular interactions of the DnaA protein are 6252 Streptomyces DnaA 3–20 times weaker than the interactions between DnaA protein course of Streptomyces evolution, the structure and the se- and its DNA target (Table IV). Probably, it facilitates effective quence of oriC region has been changed. interactions of the DnaA molecules with DNA containing mul- The relatively low affinity of Streptomyces DnaA protein for tiple recognition sequences (e.g. within the oriC region) and a single Streptomyces DnaA box seems to be compensated by a further formation of the nucleoprotein complex. For E. coli high number of DnaA boxes that are bound in a cooperative DnaA, the N-terminal domain 1 has been shown to promote manner. In comparison with the lcI repressor, the cooperativ- oligomerization (25). However, as for Streptomyces DnaA, a ity parameter of the wild-type DnaA protein (40 –100) is mod- second interaction face has been postulated in domain 3 or 4 erate; lcI repressor by binding to its three operator sites shows (26). cooperativity in the range of 250 –950 (20). Such high values in Why Does the Streptomyces oriC Region Contain so Many a system with 19 binding sites (Streptomyces oriC) might cause DnaA Boxes?—The GC content of 57 Streptomyces DnaA boxes irreversible binding of DnaA protein to the oriC region and derived from three Streptomyces oriC regions is about 10 and consequently would block subsequent replication steps. 20% lower than the GC content of the oriC region (63%) and the overall GC content of S. lividans DNA (72%), respectively. REFERENCES However, it is still significantly higher than the GC content of 1. Kornberg, A., and Baker, T. A. (1992) DNA Replication, W. H. Freeman and Co., New York the average E. coli DnaA box (;30%). This difference in GC 2. Skarstad, K., and Boye, E. (1994) Biochim. Biophys. Acta 1217, 111–130 content explains the difference in the DnaA box consensus 3. Messer, W., and Weigel, C. (1996) in Escherichia coli and Salmonella: Cellular sequence between Streptomyces (59-TTGTCCACA-39) and E. and Molecular Biology (Neidhardt, F. C., Curtiss, R., III, Ingraham, J., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, coli (59-TTATCCACA-39). It also results in flanking sequences M., and Umbarger, H. E., eds) pp. 1779 –1601, American Society for Micro- of Streptomyces DnaA boxes that are relatively rich in GC. biology, Washington, D. C. 4. Kaguni, J. M. (1997) Mol. Cells 7, 145–157 As shown in Table VII, the dissociation constant (K ) for 5. Messer, W., and Weigel, C. (1997) Mol. Microbiol. 24, 1– 6 specific binding of individual DnaA boxes derived from the S. 6. Roth, A., and Messer, W. (1995) EMBO J. 14, 2106 –2111 lividans oriC region varies between 10 and 78 nM; a few addi- 7. Schaper, S., and Messer, W. (1995) J. Biol. Chem. 270, 17622–17626 8. Speck, C., Weigel, C., and Messer, W. (1997) Nucleic Acids Res. 25, 3242–3247 tional DnaA boxes are not recognized by DnaA if they are 9. Speck, C., Weigel, C., and Messer, W. (1999) EMBO J. 18, 6169 – 6176 analyzed outside the context of oriC (K exceeding 200 nM). 10. Kutzner, H. J. (1981) in The Prokaryotes (Starr, M. P., Stolp, H., Tru ¨ per, H. G., Interestingly, the affinity of the S. lividans DnaA protein for Balows, A., and Schlegel, H. G., eds) pp. 2028 –2090 Springer, Berlin 11. Lin, Y. S., Kieser, H. M., Hopwood, D. A., and Chen, C. W. (1993) Mol. the R1/R4 E. coli DnaA box (K 5 3.4 nM)is ;3 times higher Microbiol. 10, 923–933 than its affinity for the strong Streptomyces DnaA box. The 12. Musialowski, M. S., Flett, F., Scott, G. B., Hobbs, G., Smith, C. P., and Oliver, S. G. (1994) J. Bacteriol. 176, 5123–5125 apparent dissociation constant for binding of the E. coli DnaA 13. Zakrzewska-Czerwin ˜ ska, J., and Schrempf, H. (1992) J. Bacteriol. 174, protein to the DnaA box R1/R4 was calculated to be K 5 1.1 nM 2688 –2693 (7). 14. Jakimowicz, D., Majka, J., Messer, W., Speck, C., Fernandez, M., Martin, M. C., Sanchez, J., Schauwecker, F., Keller, U., Schrempf, H., and Despite the strong difference in GC content between Strep- Zakrzewska-Czerwin ˜ ska, J. (1998) Microbiology 144, 1281–1290 tomyces and other microoganisms, including E. coli, the do- 15. Majka, J., Messer, W., Schrempf, H., and Zakrzewska-Czerwin ˜ ska, J. (1997) J. mains I and III and the binding domain of the Streptomyces Bacteriol. 179, 2426 –2432 16. Majka, J., Jakimowicz, D., Messer, W., Schrempf, H., Lisowski, M., and DnaA are highly conserved. A consequence of the high GC Zakrzewska-Czerwin ˜ ska, J. (1999) Eur. J. Biochem. 260, 325–335 content of Streptomyces structural genes (72–74%) is the very 17. Majka, J. (1997) Characterization of the Streptomyces lividans initiator protein DnaA, Ph.D. thesis, Ludwik Hirszfeld Institute of Immunology and Exper- nonrandom codon usage, with an extreme paucity of codons imental Therapy, Polish Academy of Sciences, Wroclaw, Poland with A or T in the third position (C or G is usually at the third 18. Jakimowicz, D., Majka, J., Konopa, G., Wgrzyn, G., Messer, W., Schrempf, H., codon position). Like E. coli DnaA protein, the binding domain and Zakrzewska-Czerwin ˜ ska, J. (2000) J. Mol. Biol. 298, 351–364 19. Pollock, R., and Treisman, R. (1990) Nucleic Acids Res. 18, 6197– 6204 of Streptomyces DnaA protein contains the same putative DNA 20. Senear, D. F., and Brenowitz, M. (1991) J. Biol. Chem. 266, 13661–13671 binding motive: two amphipathic a-helices with the basic loop 21. Zakrzewska-Czerwin ˜ ska, J., Nardmann, J., and Schrempf, H. (1994) Mol. Gen. in between followed by a third long a-helix (6, 16). Genet. 242, 440 – 447 22. Jakimowicz, D., Majka, J., Lis, B., Konopa, G., Wgrzyn, G., Messer, W., The affinity of the S. lividans protein for DNA fragments Schrempf, H., and Zakrzewska-Czerwin ˜ ska, J. (2000) Mol. Gen. Genet. 262, containing two or three closely spaced DnaA boxes is 6 –10 1093–1102 23. Jones, S., and Thornton, J. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13–20 times higher than its affinity for the single strong DnaA box 24. Bell, C. E., Frescura, P., Hochschild, A., and Lewis, M. (2000) Cell 101, (Table VII) and is comparable with the affinity of the E. coli 801– 811 DnaA protein to the R1/R4 box (0.9 –1.2). The data suggest that 25. Weigel, C., Schmidt, A., Seitz, H., Tuengler, D., Welzeck, M., and Messer, W. (1999) Mol. Microbiol. 34, 53– 66 efficient binding of the Streptomyces DnaA protein to DNA 26. Messer, W., Blaesing, F., Majka, J., Nardmann, J., Schaper, S., Schmidt, A., requires the presence of more than one Streptomyces DnaA box. Seitz, H., Speck, C., Tu ¨ ngler, D., We ˆ grzyn, G., Weigel, C., Welzeck, M., and Therefore, due to the high GC pressure exerted during the Zakrzewska-Czerwin ˜ ska, J. (1999) Biochimie (Paris) 81, 819 – 825

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Published: Mar 1, 2001

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