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T4 DNA ligase is more than an effective trap of cyclized dsDNA

T4 DNA ligase is more than an effective trap of cyclized dsDNA 5294–5302 Nucleic Acids Research, 2007, Vol. 35, No. 16 Published online 7 August 2007 doi:10.1093/nar/gkm582 T4 DNA ligase is more than an effective trap of cyclized dsDNA 1 1 2 2 Chongli Yuan , Xiong Wen Lou , Elizabeth Rhoades , Huimin Chen 1, and Lynden A. Archer * 1 2 School of Chemical and Biomolecular Engineering, and Department of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA Received May 16, 2007; Revised June 25, 2007; Accepted July 16, 2007 nearly all DNA-templated biological process, and can be ABSTRACT partly modulated using intrinsic regulation mechanisms T4 DNA ligase is used in standard cyclization assays that rely upon the ability of DNA sequences to create to trap double-stranded DNA (dsDNA) in low- well-ordered, reproducible nucleosome structures (2,3). probability, cyclic or highly bent conformations. In other words, the nucleosome stability, and conse- The cyclization probability, deduced from the rela- quently protein access, is likely encoded in the DNA tive yield of cyclized product, can be used in sequence itself through sequence-dependent DNA bending conjunction with statistical mechanical models to flexibility, which is inversely related to the energetic cost of forming the nucleosome particles. extract the bending stiffness of dsDNA. By inserting A variety of experimental tools have been developed to the base analog 2-aminopurine (2-AP) at designated investigate DNA bending flexibility (4–6), including positions in 89 bp and 94 bp dsDNA fragments, we comparative gel electrophoresis, crystallography, electron find that T4 DNA ligase can have a previously microscopy, transient electric birefringence and DNA unknown effect. Specifically, we observe that addi- cyclization, as well as more recent single molecule tion of T4 ligase to dsDNA in proportions compa- manipulation techniques (7–9). DNA cyclization has rable to what is used in the cyclization assay leads distinguished itself from most other approaches by its to a significant increase in fluorescence from 2-AP. simplicity, robust theoretical foundation, high sensitivity This effect is believed to originate from stabilization and accuracy, and the fact that it can be used to provide a of local base-pair opening by formation of transient simple indication of the local chain stiffness (10–13). In a DNA-ligase complexes. Non-specific binding of typical DNA cyclization experiment, DNA is designed T4 ligase to dsDNA is also confirmed using fluores- with ligatable single-stranded ends. T4 DNA ligase is added to initiate the cyclization reaction. The ratio of cence correlation spectroscopy (FCS) experiments, equilibrium constants for ligatable unimoleuclar and which reveal a systematic reduction of dsDNA bimolecular forms are measured under precise conditions diffusivity in the presence of ligase. ATP competes to determine the so-called Jacobson-Stockmayer factor, with regular DNA for non-covalent binding to the T4 J-factor. The experimentally obtained J are then fitted to ligase and is found to significantly reduce DNA- an analytical or numerical model, e.g. the continuous ligase complexation. For short dsDNA fragments, worm-like chain model (WLC) (14–19), to obtain the however, the population of DNA-ligase complexes bending flexibility of the particular sequence used. at typical ATP concentrations used in DNA cycliza- Recent cyclization data reported by Cloutier and tion studies is determined to be large enough to Widom (20,21), have produced much excitement in the dominate the cyclization reaction. literature because they indicate that short (<100 bp) double stranded DNAs (dsDNA) repeats are dramatically more flexible than anticipated by any current model for INTRODUCTION DNA bending. Marko and co-workers performed a follow-up theoretical study to explain Cloutier and In eukaryotic cells, genomic sequences are repetitively and Widom’s experimental results. These authors found that compactly packaged into nucleosomes where they are in the unusually high cyclization efficiencies reported can be close association with histone proteins. In this setting, histone proteins and DNA form tightly knit complexes reproduced by a kinkable worm like chain (KWLC) model that sterically occlude DNA from interacting with other for the DNA analyte (22–24), though the fundamental proteins (1). The stability of the nucleosome controls origin of kinks in the analytes used in the experiments *To whom correspondence should be addressed. Tel: +1 607 254 8825; Fax: +1 607 255 9166; Email: laa25@cornell.edu 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2007, Vol. 35, No. 16 5295 remains unknown. An even more recent study by 25 mg/ml BSA. The buffer conditions used here are similar Vologodskii and co-workers (25) has challenged the to those employed in the standard cyclization assay, enhanced DNA flexibility reported by Cloutier and except for the absence of ATP (21,25). To evaluate the Widom’s experiments on different grounds. These authors effect of T4 DNA ligase on 2-AP fluorescence, solutions contend that the exceptionally high cyclization efficiency containing DNA and T4 DNA ligase (New England reported is correlated with the higher than normal T4 Biolabs, MA) at various concentrations were made-up and ligase concentration used in the measurements. mixed using a gentle vortexing procedure; solutions were Yuan and co-workers (26) reported a Fluorescence stabilized at room temperature for at least 30 min prior to Resonance Energy Transfer (FRET)-based technique to the measurements. The concentration of T4 DNA ligase is measure the probability distribution of short dsDNA provided by the manufacturer in New England Biolab fragments with and without base pair mismatches. These (NEB) units. The equivalent molar concentration can be authors observed that the presence of local unpaired bases quantified using Coomassie Plus Assay (Pierce Biotech, in the dsDNA fragment leads to dramatic increases in the IL). Based on this procedure, the NEB units can be probability of highly bent, kinked states implying that converted to standard molar concentrations using the local base unpairing can produce the kinked states relation, 1 unit/ml = 0.02 nM (25%). It should be noted postulated by Marko and co-workers. that the original NEB ligase contains 200 mg/ml BSA, the Here we utilize fluorescence measurements to test a key measured ligase concentration must therefore be corrected assumption about T4 DNA ligase used in cyclization by subtracting the BSA contribution. measurements. Specifically, in cyclization measurements Characteristic diffusion times of DNA molecules in the T4 ligase is used to perform a single task. Namely, to presence of T4 ligase at various concentrations were efficiently trap low-probability, highly bent DNA con- determined using a home-built fluorescence correlation formations in which the 3 hydroxyl end of one dsDNA spectroscopy (FCS) apparatus (27–29). In a typical FCS molecule approaches the 5 phosphorylated end of the measurement, fluctuations in the fluorescence intensity are same molecule. The reports by Vologodskii et al. and by quantified by temporally autocorrelating the intensity Yuan et al. motivate a perhaps obvious question: Does T4 signal. Samples containing a fixed concentration of DNA ligase always restrain itself to the assigned task? To answer (25 nM) end-labeled with fluorescein were excited with a this question, DNA molecules containing base analog, 488 nm laser, such that only fluorescently labeled DNA which can be used as indicators of the base pair stacking molecules or their complexes are visible under the status, are used to evaluate the impact of ligase on DNA microscope. The recorded autocorrelation function G() conformation under the conditions used for cyclization was analyzed using well-known procedures (27,29). If the studies reported in references (20,21,25). sample contains only one fluorescent diffusive species, G() can be written as: MATERIALS AND METHODS 1 1=2 GðÞ¼ 1 þ 1 þ ð1Þ DNA samples N  ! D D DNA oligos with 2-AP inserted at designated positions where  is the diffusion time, N is the number of were custom synthesized by Integrated DNA technologies fluorescent molecules within focal volume, and o is the (Coraville, IA). All oligos used in the study were purified ratio of the axial/radial dimensions of the observation by the manufacturer using high performance liquid volume. The value of o was characterized using Alexa 488 chromatography (HPLC); oligo sequences are detailed in dye before the measurements, and is taken to be a the Supplementary Data section, 1a. Unless otherwise 0 0 constant for the subsequent analysis. specified, the 5 and 3 ends of the oligos are comprised of When the ligase and DNA coexists in solution, DNA- hydroxyl groups. dsDNA were produced by the standard ligase complexes contribute to G() as a second diffusive annealing procedure (26). For samples containing 2-AP, species. The autocorrelation function can therefore be the ratio of the oligo containing 2-AP to the complemen- written as, tary strand is maintained at 3:4 to minimize the relative amount of 2-AP in the non-helical states. The composition 1 1=2 of dsDNA is summarized in the Supplementary Data, GðÞ¼ f 1 þ 1 þ N  ! Section 1b. All dsDNA fragments contain mutually D,DNA D,DNA complementary 4 nt overhangs at both ends. 1 1=2 þð1  fÞ 1 þ 1 þ Fluorescence measurements D,Complex D,Complex Fluorescence excitation and emission spectra were col- ð2Þ lected using a fluorophotometer (PTI inc) with a 76 mW where f is the fraction of the total labeled DNA complexed source power, 5 nm slit width, and integration time of with ligase. 0.5 s. Quartz fluorometer cells with 3 mm path length The characteristic diffusion time for DNA ( ) (Starna Cells Inc., CA) were used for all measurements. D,DNA All spectra reported were corrected in real-time for lamp can be easily determined from FCS control experiments fluctuations. The fluorescence measurement buffer con- in which no ligase is added. In the presence of tains 50 mM Tris-HCl, 10 mM MgCl , 10 mM DTT and ligase, the autocorrelation function is analyzed with a 2 5296 Nucleic Acids Research, 2007, Vol. 35, No. 16 ‘two-diffusive-species’ model (Equation 2), in which the diffusion time of DNA ( ) is fixed at the value D,DNA obtained from the control experiment. To probe the effect of ligase concentration on DNA diffusion, DNA solutions in buffer containing ligase concentrations ranging from 500 units/ml to 4000 units/ ml were employed in the FCS experiments. It is possible that changes in the ligase concentration in the medium can in of itself alter the medium viscosity, and hence the apparent diffusivity of DNA. To quantify this effect, control experiments were performed in which the diffu- sivity of labeled dsDNA was determined in media containing BSA (MW 67 kDa) at concentrations equiva- lent to those used for the T4 ligase (MW 55.3 kDa) studies. These measurements reveal no change in the characteristic diffusion time of DNA upon addition of BSA, indicating that the ligase concentrations used in the study are low- enough that trivial changes to the medium viscosity Figure 1. Fluorescence emission spectra for E89-23 and E89-16. Inset: produced by the ligase itself can be ignored in analyzing emission spectra for 89 bp DNA containing 2-AP inserted in single DNA diffusion. stranded (oligo) and double stranded (E89-23) DNA fragments. The dsDNA concentration is 1 mM. RESULTS AND DISCUSSION on the fluorescence intensity, indicating that the global 2-AP as an indicator of DNA base pair flip-out thermal stability of the as formed dsDNA does not The nucleotide 2-AP is a fluorescent analog of guanosine contribute to the observed fluorescence increase. Figure 1 and adenosine and has been used as a site-specific probe of then simultaneously shows that even in the absence of nucleic acid structure and dynamics (30,31). Fluorescence ligase some 2-AP bases are quite exposed and the effect is emission from 2-AP can be readily excited at a wavelength dramatically larger for ‘softer’ A-T rich sequence of 320 nm, far away from any DNA absorption, or domains. absorption due to DNA-protein complexes. This feature The difference of 2-AP fluorescence within different allows selective excitation of fluorescence from the 2-AP sequence contexts can originate from basic differences base even in the presence of protein residues. Significantly, in the energy transfer mechanism between stacked bases the fluorescence emission from a 2-AP nucleotide is highly [E89-23: (AApT); E89-16: (GApT)], (36–38), as well as quenched in the stacked state, and its quantum yield from differences in the rate of spontaneous base increases approximately 10-fold in the un-stacked state un-pairings, the so-called base flip-out rate. The relatively (32,33). Local conformational changes, especially stacking low base pairing and stacking energies within the AT rich status changes, can therefore be assessed from the region [the stacking energy difference between E89-23 and fluorescence of 2-AP, i.e. the 2-AP nucleotide manifests E89-16 is estimated to be around 0.2  0.3 kcal/mol for enhanced fluorescence intensity when assuming an extra regular AA and GA dinucleotides, (39)], have been argued helical conformation (see inset of Figure 1) (32–37). By to make it easier for a single nucleotide to bypass the selectively exciting the sample at 320 nm, the fluorescence energetic barrier, and spontaneously un-pair with its intensity increase can be used to report the population of complement (39–41). It has been demonstrated through spontaneously ‘flipped-out’ bases. extensive studies, that the fluorescence of 2-AP can be To demonstrate the sensitivity of these measurements, used as a sensitive reporter of the enhanced base pair dsDNA containing 2-AP inserted in different sequence un-pairing that would result from such events (31–34). contexts were synthesized. In the E89-23 sample (see Supplementary Data 1a), 2-AP is inserted in an AT rich T4 DNA ligase and its effect on DNA conformation region, while for E89-16 2-AP is located within a random Local base pair flip-out or local defects in short dsDNA genomic sequence context. The fluorescence emission fragments can contribute significantly to the measured spectra collected when the two DNA samples, at a concentration of 1.0 mM DNA in buffer, are excited at J-factors deduced from cyclization measurements. Here 320 nm are reported in Figure 1. The background emission we wish to determine what influence, if any, other species was independently measured using the neat buffer (i.e. in used in these measurements might have on such defects, the absence of DNA), and subtracted from the sample and thereby the apparent bending stiffness reported by the spectra. It is apparent from the figure that dsDNA measurements. The ATP dependent ligation reaction containing 2-AP in the AT rich region (E89-23) exhibits involves three principal steps: (i) Activation of the substantially higher fluorescence than the sample where enzyme through formation of a covalent protein-AMP 2-AP is inserted in the random sequence context (E89-16). intermediate, accompanied by release of PPi. (ii) Transfer Increasing the complementary oligo concentration, which of the nucleotide to a phosphorylated 5 -end of the nick 0 0 does not contain the 2-AP insertion, has virtually no effect or the sticky end to produce an inverted (5 )-(5 ) Nucleic Acids Research, 2007, Vol. 35, No. 16 5297 phyrophosphate bridge structure. (iii) Catalysis of the (a) transesterification reaction resulting in the joining of the nick and release of free AMP (42,43). Three components are essential for the successful and timely completion of a ligation reaction, namely, the 5 phosphorylated end and the presence of ATP and ligase. By controlling the relative abundance of these three elements, the effect of ligase on the DNA conformation at different stages of the reaction can be clarified. For these experiments, the same two 89 bp dsDNA, containing 2-AP as in the previous experiments is used. The 5 ends of the dsDNA are unphosphorylated to prevent ligation, so the molecular size remains unchanged during the measurement. Fluorescence measurements performed using the buffer containing an equal concen- tration of ligase are used to quantify any background effects, which are subtracted from the raw spectra to isolate the effect of T4 ligase concentration on DNA (b) fluorescence. Figure 2a illustrates the effect of increasing the amount of T4 DNA ligase on the fluorescence emission intensity of E89-23. DNA concentration is maintained constant, while the ligase concentration is gradually increased. Background effects are subtracted from all spectra using the procedure outlined in the last section. The spectra shown are averages from at least two runs. It is apparent from the data that as the ligase concentration is increased the fluorescence of 2-AP manifests a corresponding increase. To confirm our findings, excitation spectra are independently measured on the same DNA samples containing varying concentration of ligase. The DNA solution without ligase is treated as background during these measurements, and the final spectra are obtained by subtracting the respective background spectra from the Figure 2. (a) Fluorescence emission spectra for E89-23 at various ligase measured sample excitation spectra. The excitation concentrations. A fixed excitation wavelength (320 nm) is used spectra collected at a fixed emission wavelength of throughout. Emission spectra for DNA-free buffer solutions containing 370 nm, should therefore solely reflect any changes to equal amounts of ligase are taken as background and subtracted from the measured fluorescence spectra. (b) Excitation spectra of E89-23 at 2-AP fluorescence induced by ligase. Figure 2b sum- various ligase concentrations. Fluorescence emission is collected at a marizes the main results. It is apparent from fixed wavelength of 370 nm, and buffer solutions containing equal this figure that with increased ligase concentration, the amounts of DNA is taken as the background and subtracted from the protein excitation peak at 280 nm increases, as expected. measured spectra. However, the secondary excitation peak centered at around 310 nm, which originates from direct excitation of 2-AP also changes from insignificant to significant with revealing enhanced dsDNA flexibility. It must be kept in increasing ligase concentration. This effect has been mind, however, that the DNA concentration employed in confirmed using a larger DNA fragment, 94 bp (E94-25), our measurements is 100–1000-fold larger than those indicating that the observation is quite general. Control normally used in cyclization experiments, and that the experiments using 2-AP inserts in single-stranded DNA ligase concentration is substantially higher than the indicate that T4 ligase does not induce any changes in 100 units/ml value recommended by Du et al.as a 2-AP fluorescence in single stranded DNA. This last standard example for studies of very short DNA. observation is significant because it eliminates the trivial Implications of both effects (higher DNA concentration possibility that the presence of ligase might change some and higher than recommended ligase concentration) will bulk property of the medium (e.g. its dielectric constant) be addressed in detail in subsequent sections of the article. and thereby alter the quantum yield of 2-AP. The relative enhancement in base pair flip-out, or These observations can be interpreted either in terms of equivalently the increase in the population of DNA a higher base pair flip-out rate induced by the ligase or in containing local defects, can be directly related to the terms of an increase in the population of dsDNA increase in the fluorescence emission intensity (Figure 3). containing extra helical 2-AP nucleotides induced by the Since the quantum yield of 2-AP in its non-stacking status presence of DNA ligase. Either mechanism can in is almost 10 times higher than in the stacked status, the principle provide a physical source for the ‘kinked’ states postulated by Marko and co-workers (22–24), and as such relative amount of DNA with ligase induced 2-AP are tempting candidates to explain cyclization data flip-out, i.e. base pair flip-out, can be estimated to be 5298 Nucleic Acids Research, 2007, Vol. 35, No. 16 Figure 4. Fluorescence emission spectra of E89-23p at various ligase concentrations, fluorescence are excited at 320 nm. Emission spectra for Figure 3. Enhancement of 2-AP fluorescence emission as a function of buffer solutions containing equal amounts of ligase are taken as ligase concentration; [DNA] = 1 mM. background and subtracted from the measured spectra. around one-tenth of the fluorescence emission intensity increase, i.e. the fractional increase referenced to the zero- ligase control. The observed ligase-induced fluorescence enhancement evidently does not exhibit a significant length dependence, which implies that it originates from the general destabilization of bases along the DNA backbone. From the slope of the fitted line in Figure 3, it is also possible to estimate that the enzyme binds to the 2-AP site or 2-AP containing region with K  50 mM. The apparent K for free state ATP, which is structurally very similar to the 2-AP nucleotide, in forming the non- convalent ligase-ATP complex is reported to be below 0.15 mM. The nearly three orders difference in K means that the inserted 2-AP nucleotide is well incorporated inside the helix. Results from other experiments using a dsDNA sample in which the 5 end of the dsDNA is phosphorylated (E89-23p), but in the absence of ATP to prevent further ligation are presented in Figure 4. The results for E89-23p Figure 5. Emission spectra for E89-23 at various ATP concentrations, show similar increases in fluorescence intensity from Fluorescence are excited at 320 nm. Emission spectra for buffer dsDNA (E89-23p) as the ligase concentration is gradually solutions containing equal amounts of DNA and ATP are taken as increased. A noteworthy feature of all of these experi- background and subtracted from the measured spectra. ments is that as [Ligase] increases, the enhancement in 2-AP fluorescence seen in the presence of ligase becomes larger. reveal no measurable effect of ATP on the characteristic It is well known that to initiate the ligation reaction, fluorescence excitation or emission spectra. Addition of ATP to dsDNA solutions in buffers ATP must be present in the reaction buffer as an essential cofactor. Specifically, ATP is known to be essential for containing T4 ligase produces a very different effect. triggering the first step of the ligation reaction by forming This effect is illustrated in Figure 5 where the effect of a transient AMP-ligase complex that can bind non- ATP concentration on ligase-enhanced 2-AP fluorescence specifically to the DNA target. Also, ATP has been is shown. To avoid the potential complication of documented as a fluorescence quencher of 2-AP. molecular size change due to the ligation, E89-23 which 0 0 However, for the low ATP concentrations used in this has hydroxyl groups on both 5 and 3 ends are used for study, the characteristic emission of the 2-AP is antici- these measurements. The fluorescence background is pated to be nearly unaffected in the presence of ATP (38). obtained using buffer solutions containing the same Fluorescence measurements performed by simply adding concentration of DNA and ATP and subtracted from ATP to dsDNA with zero ligase concentration, in fact the sample spectra. It is apparent from Figure 5 that the Nucleic Acids Research, 2007, Vol. 35, No. 16 5299 addition of ATP to E89-23 solutions containing ligase gradually reverses the effect of the ligase on 2-AP fluorescence. The crystal structure of DNA ligases have been studied by several groups (45–47). These studies reveal highly modular structures, all with preserved nucleotide-binding domains or an oligomer-binding (OB) fold. This DNA binding domain is generally positively charged and is thus capable of stabilizing the dsDNA molecules containing transiently unpaired bases by binding non-specifically to the DNA. The ATP binding domain is generally postulated to be close to the DNA binding domain (45). The diminishing ‘ligase-enhanced’ fluorescence observed after ATP addition suggests that ATP competes with DNA for binding to the ligase. Two plausible mechanisms could explain the observed phenomena. One is that after ATP binds to the ligase, it triggers a conformational change of the protein and thus precludes the ligase from acting as a stabilizing agent for spontaneously unpaired Figure 6. Relative ratio of DNA-ligase complex to DNA as a function bases in the dsDNA fragments. The other is that ATP of ligase concentration determined by analyzing the autocorrelation binding enhanced the specific binding of ligase towards its function using two diffusing species. Inset: characteristic diffusion time end and thus makes the internal binding on DNA of DNA, obtained by analyzing the autocorrelation function using a backbone less favorable. single diffusive species model, as a function of ligase concentration. Effect of T4 DNA ligase on diffusivity of DNA the basis of the 2-AP fluorescence data. We emphasize here that the DNA specimen used for FCS measurements From the emission spectra of 2-AP incorporated inside do not contain 2-AP, demonstrating that the insertion of DNA, it already seems clear that T4 DNA ligase is 2-AP in the previous fluorescence experiments does not capable of enhancing the 2-AP base flip out, although create preferential binding site for the ligase. Also, 2-AP is generally well incorporated inside the double helix essentially identical results are obtained in similar experi- and does not affect the conformation of B-type DNA. ments using 89 bp DNA sample without 2-AP insertion or It nonetheless remains unclear whether the existence of 4 nt dangling ends, which indicates that T4 DNA ligase this transient DNA-ligase complex is, not only demon- does not preferentially bind to the dangling end of the strated but also, somehow triggered by the 2-AP DNA. incorporation. To answer this question, we performed More careful scrutiny of the fluorescence correlation independent FCS measurements to determine what effect, function indicates that addition of ligase does not if any, T4 DNA ligase has on the transport properties of uniformly slow down diffusion of the DNA. Specifically, the dsDNA fragments without the 2-AP insertion. If, as by fitting the correlation function data to a model for two argued above, T4 ligase enhances base-pair flip out by diffusing species, the ligase is found instead to produce a non-specific binding to dsDNA, formation of the postu- second slow-diffusing DNA (only DNA is fluorescently lated DNA-ligase complex should be reflected as a labeled) component with a slower characteristic time reduction in the translation diffusivity of the dsDNA , that increases as the ligase concentration is fragments measured in the presence of ligase. This effect D;slow increased. Significantly, even in buffers with high ligase should be above and beyond any trivial reduction of DNA concentration, the two species model indicates that there is diffusivity produced by the ligase-induced enhancement of always a significant population of DNA that remains the bulk viscosity of the medium. effectively unaffected by the ligase (i.e.    , FCS measurements were performed at 258C using a D;fast D;DNA irrespective of [Ligase]). fluorescently labeled 89 bp non-phosphorylated DNA, Considering the low concentration of DNA used for the without 2-AP insertion. The inset in Figure 6 illustrates measurements the relative abundance of the slower, the effect of ligase concentration on the characteristic presumably ligase-complexed, species can be estimated diffusion time deduced from the FCS data by fitting the as the relative ratio of slow diffusion (f) species. Figure 6 fluorescence correlation function for a single diffusing shows the effect of ligase concentration on f calculated in species. It is evident from the figure that the average this manner. The slope of the straight-line fits to the data diffusion time of the DNA is, significantly increased by the is inversely related toK , the dissociation constant of the addition of ligase. Furthermore, as the concentration of DNA-ligase complex. At the lowest ligase concentration ligase is increased, the diffusion time at first increases quite (i.e. in the range where DNA bending studies are strongly but then manifests a weaker but steady increase performed using cyclization) we find K  1.5 mM, while with ligase concentration, over nearly four decades of concentration. The apparent ligase-induced slowing down at higher ligase concentrations we obtain K  15 mM. of DNA diffusion is consistent, at least qualitatively, with These values are, respectively, 30-times and 3-times formation of the DNA-ligase complexes hypothesized on smaller than the K value calculated previously using the d 5300 Nucleic Acids Research, 2007, Vol. 35, No. 16 2-AP fluorescence data. Taken at face value, they suggest with 1 mM ATP, as in a typical ligation buffer 8 7 that the DNA-ligase complexes are most stable at the [K = 0.15 mM, (39–40)], only 10 –10 d,ATP lowest ligase concentrations. These conclusions obviously ðK ½Ligase=K ½ATPÞ DNA contains the induced d,ATP overlook the complicating influence of multiple ligase defects. This is a very small population of the DNA molecules binding to a single DNA fragment. Specifically, molecules, and its effect generally negligible when the K as calculated previously, reflects the dissociation length of DNA is much larger than its persistence length. constant when ligase is bound ‘specifically’ to the 2-AP However, as the length of DNA decreases, the population site or the 2-AP incorporated regions. To take into of cyclizable species reduces dramatically and finally account the possibility of multiple binding sites on a single approaches and becomes even smaller than this value. substrate, relative abundance data are conventionally For example for the 89 bp DNA used in reference (15) analyzed using the Hill Function, p = [Ligase] / the J-factor predicted by the WLC model, with a (K + [Ligase] ).The solid line in Figure 6, is obtained by persistence length of 50 nm, is 10 M, which is equiva- 12 11 fitting this functional form to the experimental data. The lent to 10 –10 of the total DNA molecules. Since fit is evidently not as good as the dual straight line fit, DNA molecules containing local defects stabilized by particularly at low [Ligase]. We nonetheless proceed to ligase are expected to have a much higher tendency to be extract values of n  0.3  0.1 and K  (5  16)  10 from cyclized, the contribution to the J-factor measured in the the Hill function fit to the data. A Hill coefficient, n, less cyclization assay is clearly very important even at the ATP than unity implies that the binding event is negatively concentrations currently employed. cooperative, while the value of K has less physical meaning To provide accurate measurements of DNA stiffness both due to its large uncertainty and the lack of a direct using cyclization, our results in fact indicate that for a correlation with the binding constant for the reaction. fixed concentration of ligase, the relative ratio of ATP and Based on both analyses, we therefore conclude that ligase DNA must be carefully tuned to prevent non-specific has a tendency to bind non-specifically to the DNA and to binding of ligase to DNA. However, since ATP is form a transiently stable complex with a dissociation generally consumed during the ligation reaction, reaction constant estimated to be 2 – 25 mM, depending on the time and substrate concentration should also be taken into ligase concentration. consideration while selecting the optimal ATP concentra- It must be noted that the FCS experiments are carried tion. To illustrate, consider the cyclization measurements out under conditions with higher ligase concentration as conducted on 105 bp DNA by Du and co-workers (25). well as with a reaction buffer without added ATP as These authors managed to recover the cyclization compared with typical cyclization reaction. The higher efficiency predicted by WLC model for 106 bp DNA, ligase concentration is essential for resolving the slow using 25 units/ml ligase, 0.025 nM DNA and 1 mM ATP diffusive species, and at lowest ligase concentration being (25). Under these conditions, the relative abundance of 10 9 explored, less than 1% of DNA forms complex by binding DNA-ligase complexes is calculated to be 10 –10 , to the ligase. The effect of ATP is also examined in the compared with 10 cyclizable DNA relative to all FCS measurement. With 1 mM ATP present in the DNA molecules present. It is then possible to estimate reaction buffer, the binding of the ligase to the DNA that either an ATP concentration of over 10 M or ligase molecules is effectively suppressed, exhibiting constant concentration <10 units/ml is needed to fully suppress diffusion times independent of the added ligase concentra- the contribution of ligase induced DNA destabilization to tion as indicated in the inset of Figure 6. the measured J-factor. Our observations therefore appear to provide a plausible answer to the longstanding controversy as to Implications for DNA cyclization reactions the variability of bending stiffness values for short DNA The findings reported in the last section suggest that by fragments reported by various groups (20,21,25). stabilizing transiently unpaired bases, T4 ligase can Specifically, our results indicate that the presence of dramatically lower the bending stiffness of DNA. This large amounts of ligase in cyclization experiments might effect is analogous to that produced local defects, e.g. base well create rather than simply trap highly bent DNA pair mismatches, gaps and bulges, on the bending stiffness conformations; implying that the cyclization assay may be of DNA (26,48–49). However, we have already reported inherently not suitable for extracting bending stiffness of that ATP can suppress such non-specific binding of ligase very short DNA fragments. to the DNA backbone, suggesting that the effect can be reversed by employing a sufficiently large ATP concentra- tion in cyclization measurements. To further evaluate this CONCLUSIONS possibility, we now employ the thermodynamic constants Using the base analog 2-AP, whose fluorescence is obtained in the last section to determine the effect of ATP on the population of DNA containing defects. We begin sensitive to its microenvironment, the local conforma- by setting the ligase concentration to be around 100–400 tional change of DNA molecule under standard condi- units/ml (2–8 nM), as commonly used in the cyclization tions of the cyclization assay is tested. Existence of T4 4 3 assay. At zero ATP concentration, 10 –10 DNA ligase enhanced 2-AP fluorescence indicates that (½Ligase=K ) DNA contains local defects induced and/or ligase is capable of inducing DNA conformation change. stabilized by ligase binding. As the ATP concentration The non-specific interaction between DNA and ligase increases, the relative percentage decreases. For example, increases the subpopulation of DNA molecules containing Nucleic Acids Research, 2007, Vol. 35, No. 16 5301 10. Crothers,D.M., Drak,J., Kahn,J.D. and Levene,S.D. (1992) DNA local defects, i.e. extra-helical nucleotides, which can lower bending, flexibility and helical repeat by cyclization kinetics. the apparent stiffness of DNA determined from cycliza- Methods Enzymol., 212, 3–29. tion data. The presence of ATP can reduce this ‘ligase 11. Shore,D., Langowski,J. and Baldwin,R.L. (1981) DNA flexibility enhanced’ fluorescence by competitively binding to the studied by covalent closure of short fragments into circles. PNAS, 78, 4833–4837. functional domain of ligase and thus suppress the DNA 12. Shimada,J. and Yamakawa,H. (1984) Ring-closure probabilities for conformational change due to the non-specific DNA twisted wormlike chains-application to DNA. Macromolecules, 17, ligase contact. 689–698. In performing a meaningful cyclization assay, the ligase 13. Zhang,Y. and Crothers,D.M. (2003) Statistical mechanics of and ATP concentration must be selected with extra sequence-dependent circular DNA and its application for DNA cyclization. Biophysical J., 84, 136–153. caution. Besides satisfying the kinetic assumption under- 14. Benham,C.J. (1977) Elastic model of supercoiling. PNAS, 74, lying the J analysis (25), the ligase induced DNA 2397–2401. conformational change must be suppressed efficiently. 15. Hao,M.H. and Olson,W.K. (1989) Global equilibrium- Performing the reaction in an ATP rich medium seems to configurations of supercoiled DNA. Macromolecules, 22, 3292–3303. 16. Bauer,W.R., Lund,R.A. and White,J.H. (1993) Twist and writhe of be a promising resolution to this problem. But as DNA a DNA loop containing intrinsic bends. PNAS, 90, 833–837. size reduces, sufficiently high ligase concentration is 17. Balaeff,A., Mahadevan,L. and Schulten,K. (1999) Elastic rod model essential for efficient completion of ligation reaction and of a DNA loop in the LAC operon. Phys. Rev. Lett., 83, the preference of DNA with local defects in the cyclization 4900–4903. reaction can shift the binding competition between ATP 18. Yang,Y., Tobias,I. and Olson,W.K. (1993) Finite element analysis of DNA supercoiling. J. Chem. Phys., 98, 1673–1686. and DNA towards the DNA side. All of these factors 19. Yang,Y., Westcotts,T.P., Pedersen,S.C., Tobias,I. and Olson,W.K. might contribute to the complexity in analyzing the (1995) Effects of localized bending on DNA supercoiling. Trends cyclization data of short DNA strands and a measurement Biochem. Sci., 20, 313–319. of DNA bending stiffness without external agent could be 20. Cloutier,T.E. and Widom,J. (2004) Spontaneous sharp bending of double-stranded DNA. Mol. Cell, 14, 355–362. required in resolving this issue. 21. Cloutier,T.E. and Widom,J. (2005) DNA twisting flexibility and the formation of sharply looped protein-DNA complex. PNAS, 102, 3645–3650. SUPPLEMENTARY DATA 22. Yan,J. and Marko,J.F. (2004) Localized single-stranded bubble mechanism for cyclization of short double helix DNA. Phys. Rev. Supplementary Data are available at NAR Online. Lett., 93, 108108. 23. Wiggins,P.A., Phillips,R. and Nelson,P.C. (2005) Exact theory of kinkable elastic polymers. Phys. Rev. E., 71, 021909. 24. Yan,J., Kawamura,R. and Marko,J.F. (2005) Statistics of loop ACKNOWLEDGEMENTS formation along double helix DNAs. Phys. Rev. E., 71, 061905. We are grateful to the National Science Foundation (grant 25. Du,Q., Smith,C., Shiffeldrim,N., Vologodskaia,M. and Vologodskii,A. (2005) Cyclization of short DNA fragments DMR0551185) for supporting this study. Funding to pay and bending fluctuations of the double helix. PNAS, 102, the Open Access publication charges for this article was 5397–5402. provided by National Science Foundation (Grant DMR 26. Yuan,C., Rhoades,E., Lou,X.W. and Archer,L.A. (2006) 0551185). Spontaneous sharp bending of DNA: Role of melting bubbles. Nucleic Acids Res., 34, 4554–4560. Conflict of interest statement: None declared. 27. Rhoades,E., Ramlall,T.F., Webb,W.W. and Eliezer,D. (2006) Quantification of a-Synuclein binding to lipid vesicles using fluorescence correlation spectroscopy. Biophys. J., 90, 4692–4700. 28. Park,H.Y., Qiu,X., Rhoades,E., Korlach,J., Kwok,L.W., REFERENCES Zipfel,W.R., Webb,W.W. and Pollack,L. (2006) Achieving uniform 1. Richmond,T.J. and Davey,C.A. (2003) The structure of DNA in the mixing in microfluidic device: hydrodynamic focusing prior to mixing. Analytical Chem., 78, 4465–4473. nuclesome core. 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T4 DNA ligase is more than an effective trap of cyclized dsDNA

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Abstract

5294–5302 Nucleic Acids Research, 2007, Vol. 35, No. 16 Published online 7 August 2007 doi:10.1093/nar/gkm582 T4 DNA ligase is more than an effective trap of cyclized dsDNA 1 1 2 2 Chongli Yuan , Xiong Wen Lou , Elizabeth Rhoades , Huimin Chen 1, and Lynden A. Archer * 1 2 School of Chemical and Biomolecular Engineering, and Department of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA Received May 16, 2007; Revised June 25, 2007; Accepted July 16, 2007 nearly all DNA-templated biological process, and can be ABSTRACT partly modulated using intrinsic regulation mechanisms T4 DNA ligase is used in standard cyclization assays that rely upon the ability of DNA sequences to create to trap double-stranded DNA (dsDNA) in low- well-ordered, reproducible nucleosome structures (2,3). probability, cyclic or highly bent conformations. In other words, the nucleosome stability, and conse- The cyclization probability, deduced from the rela- quently protein access, is likely encoded in the DNA tive yield of cyclized product, can be used in sequence itself through sequence-dependent DNA bending conjunction with statistical mechanical models to flexibility, which is inversely related to the energetic cost of forming the nucleosome particles. extract the bending stiffness of dsDNA. By inserting A variety of experimental tools have been developed to the base analog 2-aminopurine (2-AP) at designated investigate DNA bending flexibility (4–6), including positions in 89 bp and 94 bp dsDNA fragments, we comparative gel electrophoresis, crystallography, electron find that T4 DNA ligase can have a previously microscopy, transient electric birefringence and DNA unknown effect. Specifically, we observe that addi- cyclization, as well as more recent single molecule tion of T4 ligase to dsDNA in proportions compa- manipulation techniques (7–9). DNA cyclization has rable to what is used in the cyclization assay leads distinguished itself from most other approaches by its to a significant increase in fluorescence from 2-AP. simplicity, robust theoretical foundation, high sensitivity This effect is believed to originate from stabilization and accuracy, and the fact that it can be used to provide a of local base-pair opening by formation of transient simple indication of the local chain stiffness (10–13). In a DNA-ligase complexes. Non-specific binding of typical DNA cyclization experiment, DNA is designed T4 ligase to dsDNA is also confirmed using fluores- with ligatable single-stranded ends. T4 DNA ligase is added to initiate the cyclization reaction. The ratio of cence correlation spectroscopy (FCS) experiments, equilibrium constants for ligatable unimoleuclar and which reveal a systematic reduction of dsDNA bimolecular forms are measured under precise conditions diffusivity in the presence of ligase. ATP competes to determine the so-called Jacobson-Stockmayer factor, with regular DNA for non-covalent binding to the T4 J-factor. The experimentally obtained J are then fitted to ligase and is found to significantly reduce DNA- an analytical or numerical model, e.g. the continuous ligase complexation. For short dsDNA fragments, worm-like chain model (WLC) (14–19), to obtain the however, the population of DNA-ligase complexes bending flexibility of the particular sequence used. at typical ATP concentrations used in DNA cycliza- Recent cyclization data reported by Cloutier and tion studies is determined to be large enough to Widom (20,21), have produced much excitement in the dominate the cyclization reaction. literature because they indicate that short (<100 bp) double stranded DNAs (dsDNA) repeats are dramatically more flexible than anticipated by any current model for INTRODUCTION DNA bending. Marko and co-workers performed a follow-up theoretical study to explain Cloutier and In eukaryotic cells, genomic sequences are repetitively and Widom’s experimental results. These authors found that compactly packaged into nucleosomes where they are in the unusually high cyclization efficiencies reported can be close association with histone proteins. In this setting, histone proteins and DNA form tightly knit complexes reproduced by a kinkable worm like chain (KWLC) model that sterically occlude DNA from interacting with other for the DNA analyte (22–24), though the fundamental proteins (1). The stability of the nucleosome controls origin of kinks in the analytes used in the experiments *To whom correspondence should be addressed. Tel: +1 607 254 8825; Fax: +1 607 255 9166; Email: laa25@cornell.edu 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2007, Vol. 35, No. 16 5295 remains unknown. An even more recent study by 25 mg/ml BSA. The buffer conditions used here are similar Vologodskii and co-workers (25) has challenged the to those employed in the standard cyclization assay, enhanced DNA flexibility reported by Cloutier and except for the absence of ATP (21,25). To evaluate the Widom’s experiments on different grounds. These authors effect of T4 DNA ligase on 2-AP fluorescence, solutions contend that the exceptionally high cyclization efficiency containing DNA and T4 DNA ligase (New England reported is correlated with the higher than normal T4 Biolabs, MA) at various concentrations were made-up and ligase concentration used in the measurements. mixed using a gentle vortexing procedure; solutions were Yuan and co-workers (26) reported a Fluorescence stabilized at room temperature for at least 30 min prior to Resonance Energy Transfer (FRET)-based technique to the measurements. The concentration of T4 DNA ligase is measure the probability distribution of short dsDNA provided by the manufacturer in New England Biolab fragments with and without base pair mismatches. These (NEB) units. The equivalent molar concentration can be authors observed that the presence of local unpaired bases quantified using Coomassie Plus Assay (Pierce Biotech, in the dsDNA fragment leads to dramatic increases in the IL). Based on this procedure, the NEB units can be probability of highly bent, kinked states implying that converted to standard molar concentrations using the local base unpairing can produce the kinked states relation, 1 unit/ml = 0.02 nM (25%). It should be noted postulated by Marko and co-workers. that the original NEB ligase contains 200 mg/ml BSA, the Here we utilize fluorescence measurements to test a key measured ligase concentration must therefore be corrected assumption about T4 DNA ligase used in cyclization by subtracting the BSA contribution. measurements. Specifically, in cyclization measurements Characteristic diffusion times of DNA molecules in the T4 ligase is used to perform a single task. Namely, to presence of T4 ligase at various concentrations were efficiently trap low-probability, highly bent DNA con- determined using a home-built fluorescence correlation formations in which the 3 hydroxyl end of one dsDNA spectroscopy (FCS) apparatus (27–29). In a typical FCS molecule approaches the 5 phosphorylated end of the measurement, fluctuations in the fluorescence intensity are same molecule. The reports by Vologodskii et al. and by quantified by temporally autocorrelating the intensity Yuan et al. motivate a perhaps obvious question: Does T4 signal. Samples containing a fixed concentration of DNA ligase always restrain itself to the assigned task? To answer (25 nM) end-labeled with fluorescein were excited with a this question, DNA molecules containing base analog, 488 nm laser, such that only fluorescently labeled DNA which can be used as indicators of the base pair stacking molecules or their complexes are visible under the status, are used to evaluate the impact of ligase on DNA microscope. The recorded autocorrelation function G() conformation under the conditions used for cyclization was analyzed using well-known procedures (27,29). If the studies reported in references (20,21,25). sample contains only one fluorescent diffusive species, G() can be written as: MATERIALS AND METHODS 1 1=2 GðÞ¼ 1 þ 1 þ ð1Þ DNA samples N  ! D D DNA oligos with 2-AP inserted at designated positions where  is the diffusion time, N is the number of were custom synthesized by Integrated DNA technologies fluorescent molecules within focal volume, and o is the (Coraville, IA). All oligos used in the study were purified ratio of the axial/radial dimensions of the observation by the manufacturer using high performance liquid volume. The value of o was characterized using Alexa 488 chromatography (HPLC); oligo sequences are detailed in dye before the measurements, and is taken to be a the Supplementary Data section, 1a. Unless otherwise 0 0 constant for the subsequent analysis. specified, the 5 and 3 ends of the oligos are comprised of When the ligase and DNA coexists in solution, DNA- hydroxyl groups. dsDNA were produced by the standard ligase complexes contribute to G() as a second diffusive annealing procedure (26). For samples containing 2-AP, species. The autocorrelation function can therefore be the ratio of the oligo containing 2-AP to the complemen- written as, tary strand is maintained at 3:4 to minimize the relative amount of 2-AP in the non-helical states. The composition 1 1=2 of dsDNA is summarized in the Supplementary Data, GðÞ¼ f 1 þ 1 þ N  ! Section 1b. All dsDNA fragments contain mutually D,DNA D,DNA complementary 4 nt overhangs at both ends. 1 1=2 þð1  fÞ 1 þ 1 þ Fluorescence measurements D,Complex D,Complex Fluorescence excitation and emission spectra were col- ð2Þ lected using a fluorophotometer (PTI inc) with a 76 mW where f is the fraction of the total labeled DNA complexed source power, 5 nm slit width, and integration time of with ligase. 0.5 s. Quartz fluorometer cells with 3 mm path length The characteristic diffusion time for DNA ( ) (Starna Cells Inc., CA) were used for all measurements. D,DNA All spectra reported were corrected in real-time for lamp can be easily determined from FCS control experiments fluctuations. The fluorescence measurement buffer con- in which no ligase is added. In the presence of tains 50 mM Tris-HCl, 10 mM MgCl , 10 mM DTT and ligase, the autocorrelation function is analyzed with a 2 5296 Nucleic Acids Research, 2007, Vol. 35, No. 16 ‘two-diffusive-species’ model (Equation 2), in which the diffusion time of DNA ( ) is fixed at the value D,DNA obtained from the control experiment. To probe the effect of ligase concentration on DNA diffusion, DNA solutions in buffer containing ligase concentrations ranging from 500 units/ml to 4000 units/ ml were employed in the FCS experiments. It is possible that changes in the ligase concentration in the medium can in of itself alter the medium viscosity, and hence the apparent diffusivity of DNA. To quantify this effect, control experiments were performed in which the diffu- sivity of labeled dsDNA was determined in media containing BSA (MW 67 kDa) at concentrations equiva- lent to those used for the T4 ligase (MW 55.3 kDa) studies. These measurements reveal no change in the characteristic diffusion time of DNA upon addition of BSA, indicating that the ligase concentrations used in the study are low- enough that trivial changes to the medium viscosity Figure 1. Fluorescence emission spectra for E89-23 and E89-16. Inset: produced by the ligase itself can be ignored in analyzing emission spectra for 89 bp DNA containing 2-AP inserted in single DNA diffusion. stranded (oligo) and double stranded (E89-23) DNA fragments. The dsDNA concentration is 1 mM. RESULTS AND DISCUSSION on the fluorescence intensity, indicating that the global 2-AP as an indicator of DNA base pair flip-out thermal stability of the as formed dsDNA does not The nucleotide 2-AP is a fluorescent analog of guanosine contribute to the observed fluorescence increase. Figure 1 and adenosine and has been used as a site-specific probe of then simultaneously shows that even in the absence of nucleic acid structure and dynamics (30,31). Fluorescence ligase some 2-AP bases are quite exposed and the effect is emission from 2-AP can be readily excited at a wavelength dramatically larger for ‘softer’ A-T rich sequence of 320 nm, far away from any DNA absorption, or domains. absorption due to DNA-protein complexes. This feature The difference of 2-AP fluorescence within different allows selective excitation of fluorescence from the 2-AP sequence contexts can originate from basic differences base even in the presence of protein residues. Significantly, in the energy transfer mechanism between stacked bases the fluorescence emission from a 2-AP nucleotide is highly [E89-23: (AApT); E89-16: (GApT)], (36–38), as well as quenched in the stacked state, and its quantum yield from differences in the rate of spontaneous base increases approximately 10-fold in the un-stacked state un-pairings, the so-called base flip-out rate. The relatively (32,33). Local conformational changes, especially stacking low base pairing and stacking energies within the AT rich status changes, can therefore be assessed from the region [the stacking energy difference between E89-23 and fluorescence of 2-AP, i.e. the 2-AP nucleotide manifests E89-16 is estimated to be around 0.2  0.3 kcal/mol for enhanced fluorescence intensity when assuming an extra regular AA and GA dinucleotides, (39)], have been argued helical conformation (see inset of Figure 1) (32–37). By to make it easier for a single nucleotide to bypass the selectively exciting the sample at 320 nm, the fluorescence energetic barrier, and spontaneously un-pair with its intensity increase can be used to report the population of complement (39–41). It has been demonstrated through spontaneously ‘flipped-out’ bases. extensive studies, that the fluorescence of 2-AP can be To demonstrate the sensitivity of these measurements, used as a sensitive reporter of the enhanced base pair dsDNA containing 2-AP inserted in different sequence un-pairing that would result from such events (31–34). contexts were synthesized. In the E89-23 sample (see Supplementary Data 1a), 2-AP is inserted in an AT rich T4 DNA ligase and its effect on DNA conformation region, while for E89-16 2-AP is located within a random Local base pair flip-out or local defects in short dsDNA genomic sequence context. The fluorescence emission fragments can contribute significantly to the measured spectra collected when the two DNA samples, at a concentration of 1.0 mM DNA in buffer, are excited at J-factors deduced from cyclization measurements. Here 320 nm are reported in Figure 1. The background emission we wish to determine what influence, if any, other species was independently measured using the neat buffer (i.e. in used in these measurements might have on such defects, the absence of DNA), and subtracted from the sample and thereby the apparent bending stiffness reported by the spectra. It is apparent from the figure that dsDNA measurements. The ATP dependent ligation reaction containing 2-AP in the AT rich region (E89-23) exhibits involves three principal steps: (i) Activation of the substantially higher fluorescence than the sample where enzyme through formation of a covalent protein-AMP 2-AP is inserted in the random sequence context (E89-16). intermediate, accompanied by release of PPi. (ii) Transfer Increasing the complementary oligo concentration, which of the nucleotide to a phosphorylated 5 -end of the nick 0 0 does not contain the 2-AP insertion, has virtually no effect or the sticky end to produce an inverted (5 )-(5 ) Nucleic Acids Research, 2007, Vol. 35, No. 16 5297 phyrophosphate bridge structure. (iii) Catalysis of the (a) transesterification reaction resulting in the joining of the nick and release of free AMP (42,43). Three components are essential for the successful and timely completion of a ligation reaction, namely, the 5 phosphorylated end and the presence of ATP and ligase. By controlling the relative abundance of these three elements, the effect of ligase on the DNA conformation at different stages of the reaction can be clarified. For these experiments, the same two 89 bp dsDNA, containing 2-AP as in the previous experiments is used. The 5 ends of the dsDNA are unphosphorylated to prevent ligation, so the molecular size remains unchanged during the measurement. Fluorescence measurements performed using the buffer containing an equal concen- tration of ligase are used to quantify any background effects, which are subtracted from the raw spectra to isolate the effect of T4 ligase concentration on DNA (b) fluorescence. Figure 2a illustrates the effect of increasing the amount of T4 DNA ligase on the fluorescence emission intensity of E89-23. DNA concentration is maintained constant, while the ligase concentration is gradually increased. Background effects are subtracted from all spectra using the procedure outlined in the last section. The spectra shown are averages from at least two runs. It is apparent from the data that as the ligase concentration is increased the fluorescence of 2-AP manifests a corresponding increase. To confirm our findings, excitation spectra are independently measured on the same DNA samples containing varying concentration of ligase. The DNA solution without ligase is treated as background during these measurements, and the final spectra are obtained by subtracting the respective background spectra from the Figure 2. (a) Fluorescence emission spectra for E89-23 at various ligase measured sample excitation spectra. The excitation concentrations. A fixed excitation wavelength (320 nm) is used spectra collected at a fixed emission wavelength of throughout. Emission spectra for DNA-free buffer solutions containing 370 nm, should therefore solely reflect any changes to equal amounts of ligase are taken as background and subtracted from the measured fluorescence spectra. (b) Excitation spectra of E89-23 at 2-AP fluorescence induced by ligase. Figure 2b sum- various ligase concentrations. Fluorescence emission is collected at a marizes the main results. It is apparent from fixed wavelength of 370 nm, and buffer solutions containing equal this figure that with increased ligase concentration, the amounts of DNA is taken as the background and subtracted from the protein excitation peak at 280 nm increases, as expected. measured spectra. However, the secondary excitation peak centered at around 310 nm, which originates from direct excitation of 2-AP also changes from insignificant to significant with revealing enhanced dsDNA flexibility. It must be kept in increasing ligase concentration. This effect has been mind, however, that the DNA concentration employed in confirmed using a larger DNA fragment, 94 bp (E94-25), our measurements is 100–1000-fold larger than those indicating that the observation is quite general. Control normally used in cyclization experiments, and that the experiments using 2-AP inserts in single-stranded DNA ligase concentration is substantially higher than the indicate that T4 ligase does not induce any changes in 100 units/ml value recommended by Du et al.as a 2-AP fluorescence in single stranded DNA. This last standard example for studies of very short DNA. observation is significant because it eliminates the trivial Implications of both effects (higher DNA concentration possibility that the presence of ligase might change some and higher than recommended ligase concentration) will bulk property of the medium (e.g. its dielectric constant) be addressed in detail in subsequent sections of the article. and thereby alter the quantum yield of 2-AP. The relative enhancement in base pair flip-out, or These observations can be interpreted either in terms of equivalently the increase in the population of DNA a higher base pair flip-out rate induced by the ligase or in containing local defects, can be directly related to the terms of an increase in the population of dsDNA increase in the fluorescence emission intensity (Figure 3). containing extra helical 2-AP nucleotides induced by the Since the quantum yield of 2-AP in its non-stacking status presence of DNA ligase. Either mechanism can in is almost 10 times higher than in the stacked status, the principle provide a physical source for the ‘kinked’ states postulated by Marko and co-workers (22–24), and as such relative amount of DNA with ligase induced 2-AP are tempting candidates to explain cyclization data flip-out, i.e. base pair flip-out, can be estimated to be 5298 Nucleic Acids Research, 2007, Vol. 35, No. 16 Figure 4. Fluorescence emission spectra of E89-23p at various ligase concentrations, fluorescence are excited at 320 nm. Emission spectra for Figure 3. Enhancement of 2-AP fluorescence emission as a function of buffer solutions containing equal amounts of ligase are taken as ligase concentration; [DNA] = 1 mM. background and subtracted from the measured spectra. around one-tenth of the fluorescence emission intensity increase, i.e. the fractional increase referenced to the zero- ligase control. The observed ligase-induced fluorescence enhancement evidently does not exhibit a significant length dependence, which implies that it originates from the general destabilization of bases along the DNA backbone. From the slope of the fitted line in Figure 3, it is also possible to estimate that the enzyme binds to the 2-AP site or 2-AP containing region with K  50 mM. The apparent K for free state ATP, which is structurally very similar to the 2-AP nucleotide, in forming the non- convalent ligase-ATP complex is reported to be below 0.15 mM. The nearly three orders difference in K means that the inserted 2-AP nucleotide is well incorporated inside the helix. Results from other experiments using a dsDNA sample in which the 5 end of the dsDNA is phosphorylated (E89-23p), but in the absence of ATP to prevent further ligation are presented in Figure 4. The results for E89-23p Figure 5. Emission spectra for E89-23 at various ATP concentrations, show similar increases in fluorescence intensity from Fluorescence are excited at 320 nm. Emission spectra for buffer dsDNA (E89-23p) as the ligase concentration is gradually solutions containing equal amounts of DNA and ATP are taken as increased. A noteworthy feature of all of these experi- background and subtracted from the measured spectra. ments is that as [Ligase] increases, the enhancement in 2-AP fluorescence seen in the presence of ligase becomes larger. reveal no measurable effect of ATP on the characteristic It is well known that to initiate the ligation reaction, fluorescence excitation or emission spectra. Addition of ATP to dsDNA solutions in buffers ATP must be present in the reaction buffer as an essential cofactor. Specifically, ATP is known to be essential for containing T4 ligase produces a very different effect. triggering the first step of the ligation reaction by forming This effect is illustrated in Figure 5 where the effect of a transient AMP-ligase complex that can bind non- ATP concentration on ligase-enhanced 2-AP fluorescence specifically to the DNA target. Also, ATP has been is shown. To avoid the potential complication of documented as a fluorescence quencher of 2-AP. molecular size change due to the ligation, E89-23 which 0 0 However, for the low ATP concentrations used in this has hydroxyl groups on both 5 and 3 ends are used for study, the characteristic emission of the 2-AP is antici- these measurements. The fluorescence background is pated to be nearly unaffected in the presence of ATP (38). obtained using buffer solutions containing the same Fluorescence measurements performed by simply adding concentration of DNA and ATP and subtracted from ATP to dsDNA with zero ligase concentration, in fact the sample spectra. It is apparent from Figure 5 that the Nucleic Acids Research, 2007, Vol. 35, No. 16 5299 addition of ATP to E89-23 solutions containing ligase gradually reverses the effect of the ligase on 2-AP fluorescence. The crystal structure of DNA ligases have been studied by several groups (45–47). These studies reveal highly modular structures, all with preserved nucleotide-binding domains or an oligomer-binding (OB) fold. This DNA binding domain is generally positively charged and is thus capable of stabilizing the dsDNA molecules containing transiently unpaired bases by binding non-specifically to the DNA. The ATP binding domain is generally postulated to be close to the DNA binding domain (45). The diminishing ‘ligase-enhanced’ fluorescence observed after ATP addition suggests that ATP competes with DNA for binding to the ligase. Two plausible mechanisms could explain the observed phenomena. One is that after ATP binds to the ligase, it triggers a conformational change of the protein and thus precludes the ligase from acting as a stabilizing agent for spontaneously unpaired Figure 6. Relative ratio of DNA-ligase complex to DNA as a function bases in the dsDNA fragments. The other is that ATP of ligase concentration determined by analyzing the autocorrelation binding enhanced the specific binding of ligase towards its function using two diffusing species. Inset: characteristic diffusion time end and thus makes the internal binding on DNA of DNA, obtained by analyzing the autocorrelation function using a backbone less favorable. single diffusive species model, as a function of ligase concentration. Effect of T4 DNA ligase on diffusivity of DNA the basis of the 2-AP fluorescence data. We emphasize here that the DNA specimen used for FCS measurements From the emission spectra of 2-AP incorporated inside do not contain 2-AP, demonstrating that the insertion of DNA, it already seems clear that T4 DNA ligase is 2-AP in the previous fluorescence experiments does not capable of enhancing the 2-AP base flip out, although create preferential binding site for the ligase. Also, 2-AP is generally well incorporated inside the double helix essentially identical results are obtained in similar experi- and does not affect the conformation of B-type DNA. ments using 89 bp DNA sample without 2-AP insertion or It nonetheless remains unclear whether the existence of 4 nt dangling ends, which indicates that T4 DNA ligase this transient DNA-ligase complex is, not only demon- does not preferentially bind to the dangling end of the strated but also, somehow triggered by the 2-AP DNA. incorporation. To answer this question, we performed More careful scrutiny of the fluorescence correlation independent FCS measurements to determine what effect, function indicates that addition of ligase does not if any, T4 DNA ligase has on the transport properties of uniformly slow down diffusion of the DNA. Specifically, the dsDNA fragments without the 2-AP insertion. If, as by fitting the correlation function data to a model for two argued above, T4 ligase enhances base-pair flip out by diffusing species, the ligase is found instead to produce a non-specific binding to dsDNA, formation of the postu- second slow-diffusing DNA (only DNA is fluorescently lated DNA-ligase complex should be reflected as a labeled) component with a slower characteristic time reduction in the translation diffusivity of the dsDNA , that increases as the ligase concentration is fragments measured in the presence of ligase. This effect D;slow increased. Significantly, even in buffers with high ligase should be above and beyond any trivial reduction of DNA concentration, the two species model indicates that there is diffusivity produced by the ligase-induced enhancement of always a significant population of DNA that remains the bulk viscosity of the medium. effectively unaffected by the ligase (i.e.    , FCS measurements were performed at 258C using a D;fast D;DNA irrespective of [Ligase]). fluorescently labeled 89 bp non-phosphorylated DNA, Considering the low concentration of DNA used for the without 2-AP insertion. The inset in Figure 6 illustrates measurements the relative abundance of the slower, the effect of ligase concentration on the characteristic presumably ligase-complexed, species can be estimated diffusion time deduced from the FCS data by fitting the as the relative ratio of slow diffusion (f) species. Figure 6 fluorescence correlation function for a single diffusing shows the effect of ligase concentration on f calculated in species. It is evident from the figure that the average this manner. The slope of the straight-line fits to the data diffusion time of the DNA is, significantly increased by the is inversely related toK , the dissociation constant of the addition of ligase. Furthermore, as the concentration of DNA-ligase complex. At the lowest ligase concentration ligase is increased, the diffusion time at first increases quite (i.e. in the range where DNA bending studies are strongly but then manifests a weaker but steady increase performed using cyclization) we find K  1.5 mM, while with ligase concentration, over nearly four decades of concentration. The apparent ligase-induced slowing down at higher ligase concentrations we obtain K  15 mM. of DNA diffusion is consistent, at least qualitatively, with These values are, respectively, 30-times and 3-times formation of the DNA-ligase complexes hypothesized on smaller than the K value calculated previously using the d 5300 Nucleic Acids Research, 2007, Vol. 35, No. 16 2-AP fluorescence data. Taken at face value, they suggest with 1 mM ATP, as in a typical ligation buffer 8 7 that the DNA-ligase complexes are most stable at the [K = 0.15 mM, (39–40)], only 10 –10 d,ATP lowest ligase concentrations. These conclusions obviously ðK ½Ligase=K ½ATPÞ DNA contains the induced d,ATP overlook the complicating influence of multiple ligase defects. This is a very small population of the DNA molecules binding to a single DNA fragment. Specifically, molecules, and its effect generally negligible when the K as calculated previously, reflects the dissociation length of DNA is much larger than its persistence length. constant when ligase is bound ‘specifically’ to the 2-AP However, as the length of DNA decreases, the population site or the 2-AP incorporated regions. To take into of cyclizable species reduces dramatically and finally account the possibility of multiple binding sites on a single approaches and becomes even smaller than this value. substrate, relative abundance data are conventionally For example for the 89 bp DNA used in reference (15) analyzed using the Hill Function, p = [Ligase] / the J-factor predicted by the WLC model, with a (K + [Ligase] ).The solid line in Figure 6, is obtained by persistence length of 50 nm, is 10 M, which is equiva- 12 11 fitting this functional form to the experimental data. The lent to 10 –10 of the total DNA molecules. Since fit is evidently not as good as the dual straight line fit, DNA molecules containing local defects stabilized by particularly at low [Ligase]. We nonetheless proceed to ligase are expected to have a much higher tendency to be extract values of n  0.3  0.1 and K  (5  16)  10 from cyclized, the contribution to the J-factor measured in the the Hill function fit to the data. A Hill coefficient, n, less cyclization assay is clearly very important even at the ATP than unity implies that the binding event is negatively concentrations currently employed. cooperative, while the value of K has less physical meaning To provide accurate measurements of DNA stiffness both due to its large uncertainty and the lack of a direct using cyclization, our results in fact indicate that for a correlation with the binding constant for the reaction. fixed concentration of ligase, the relative ratio of ATP and Based on both analyses, we therefore conclude that ligase DNA must be carefully tuned to prevent non-specific has a tendency to bind non-specifically to the DNA and to binding of ligase to DNA. However, since ATP is form a transiently stable complex with a dissociation generally consumed during the ligation reaction, reaction constant estimated to be 2 – 25 mM, depending on the time and substrate concentration should also be taken into ligase concentration. consideration while selecting the optimal ATP concentra- It must be noted that the FCS experiments are carried tion. To illustrate, consider the cyclization measurements out under conditions with higher ligase concentration as conducted on 105 bp DNA by Du and co-workers (25). well as with a reaction buffer without added ATP as These authors managed to recover the cyclization compared with typical cyclization reaction. The higher efficiency predicted by WLC model for 106 bp DNA, ligase concentration is essential for resolving the slow using 25 units/ml ligase, 0.025 nM DNA and 1 mM ATP diffusive species, and at lowest ligase concentration being (25). Under these conditions, the relative abundance of 10 9 explored, less than 1% of DNA forms complex by binding DNA-ligase complexes is calculated to be 10 –10 , to the ligase. The effect of ATP is also examined in the compared with 10 cyclizable DNA relative to all FCS measurement. With 1 mM ATP present in the DNA molecules present. It is then possible to estimate reaction buffer, the binding of the ligase to the DNA that either an ATP concentration of over 10 M or ligase molecules is effectively suppressed, exhibiting constant concentration <10 units/ml is needed to fully suppress diffusion times independent of the added ligase concentra- the contribution of ligase induced DNA destabilization to tion as indicated in the inset of Figure 6. the measured J-factor. Our observations therefore appear to provide a plausible answer to the longstanding controversy as to Implications for DNA cyclization reactions the variability of bending stiffness values for short DNA The findings reported in the last section suggest that by fragments reported by various groups (20,21,25). stabilizing transiently unpaired bases, T4 ligase can Specifically, our results indicate that the presence of dramatically lower the bending stiffness of DNA. This large amounts of ligase in cyclization experiments might effect is analogous to that produced local defects, e.g. base well create rather than simply trap highly bent DNA pair mismatches, gaps and bulges, on the bending stiffness conformations; implying that the cyclization assay may be of DNA (26,48–49). However, we have already reported inherently not suitable for extracting bending stiffness of that ATP can suppress such non-specific binding of ligase very short DNA fragments. to the DNA backbone, suggesting that the effect can be reversed by employing a sufficiently large ATP concentra- tion in cyclization measurements. To further evaluate this CONCLUSIONS possibility, we now employ the thermodynamic constants Using the base analog 2-AP, whose fluorescence is obtained in the last section to determine the effect of ATP on the population of DNA containing defects. We begin sensitive to its microenvironment, the local conforma- by setting the ligase concentration to be around 100–400 tional change of DNA molecule under standard condi- units/ml (2–8 nM), as commonly used in the cyclization tions of the cyclization assay is tested. Existence of T4 4 3 assay. At zero ATP concentration, 10 –10 DNA ligase enhanced 2-AP fluorescence indicates that (½Ligase=K ) DNA contains local defects induced and/or ligase is capable of inducing DNA conformation change. stabilized by ligase binding. As the ATP concentration The non-specific interaction between DNA and ligase increases, the relative percentage decreases. For example, increases the subpopulation of DNA molecules containing Nucleic Acids Research, 2007, Vol. 35, No. 16 5301 10. Crothers,D.M., Drak,J., Kahn,J.D. and Levene,S.D. (1992) DNA local defects, i.e. extra-helical nucleotides, which can lower bending, flexibility and helical repeat by cyclization kinetics. the apparent stiffness of DNA determined from cycliza- Methods Enzymol., 212, 3–29. tion data. The presence of ATP can reduce this ‘ligase 11. Shore,D., Langowski,J. and Baldwin,R.L. (1981) DNA flexibility enhanced’ fluorescence by competitively binding to the studied by covalent closure of short fragments into circles. 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Bauer,W.R., Lund,R.A. and White,J.H. (1993) Twist and writhe of be a promising resolution to this problem. But as DNA a DNA loop containing intrinsic bends. PNAS, 90, 833–837. size reduces, sufficiently high ligase concentration is 17. Balaeff,A., Mahadevan,L. and Schulten,K. (1999) Elastic rod model essential for efficient completion of ligation reaction and of a DNA loop in the LAC operon. Phys. Rev. Lett., 83, the preference of DNA with local defects in the cyclization 4900–4903. reaction can shift the binding competition between ATP 18. Yang,Y., Tobias,I. and Olson,W.K. (1993) Finite element analysis of DNA supercoiling. J. Chem. Phys., 98, 1673–1686. and DNA towards the DNA side. All of these factors 19. Yang,Y., Westcotts,T.P., Pedersen,S.C., Tobias,I. and Olson,W.K. might contribute to the complexity in analyzing the (1995) Effects of localized bending on DNA supercoiling. Trends cyclization data of short DNA strands and a measurement Biochem. 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Nucleic Acids Research – Oxford University Press

Published: Aug 7, 2007

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