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The Bloom’s syndrome helicase stimulates the activity of human topoisomerase IIIα

The Bloom’s syndrome helicase stimulates the activity of human topoisomerase IIIα ã 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30 No. 22 4823±4829 The Bloom's syndrome helicase stimulates the activity of human topoisomerase IIIa Leonard Wu and Ian D. Hickson* Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK Received August 15, 2002; Revised and Accepted September 17, 2002 ABSTRACT display a hyper-recombinogenic phenotype reminiscent of BS cells, suggesting that RecQ helicases perform a conserved Bloom's syndrome (BS) is a disorder associated function in controlling the level of homologous recombination with chromosomal instability and a predisposition in cells (14±19). Although the precise cellular role of any to the development of cancer. The BS gene product, RecQ helicase has yet to be elucidated, several lines of BLM, is a DNA helicase of the RecQ family that forms evidence suggest that RecQ helicases act in concert with type a complex in vitro and in vivo with topoisomerase IA topoisomerases (reviewed in 20). IIIa. Here, we show that BLM stimulates the ability of The type IA subclass of topoisomerases includes Escherichia coli topoisomerases I and III and the eukaryotic topoisomerase IIIa to relax negatively supercoiled topoisomerase III enzymes (reviewed in 21). In vertebrates, DNA. Moreover, DNA binding analyses indicate that there are at least two isoforms of topoisomerase III, termed a BLM recruits topoisomerase IIIa to its DNA sub- and b, which display only a weak topoisomerase activity strate. Consistent with this, a mutant form of BLM towards negatively supercoiled DNA (22±25). Yeast cells that retains helicase activity, but is unable to bind express a single topoisomerase III enzyme encoded by the topoisomerase IIIa, fails to stimulate topoisomerase TOP3 gene (26). In Saccharomyces cerevisiae, top3D mutants activity. These results indicate that a physical are viable, but grow very slowly and have defects in S phase association between BLM and topoisomerase IIIa responses to DNA damage and in both mitotic and meiotic is a prerequisite for their functional biochemical recombination (16,26±28). In contrast, the top3 gene in interaction. Schizosaccharomyces pombe is essential for viability, with top3D mutants displaying an inability to accurately segregate daughter chromosomes during mitosis (29,30). Interestingly, INTRODUCTION mutation of SGS1 or rqh1 , the sole RecQ homologues found in budding and ®ssion yeast, respectively, can suppress the Bloom's syndrome (BS) is a rare genetic disorder character- deleterious effects caused by the absence of Top3 protein ized by proportional dwar®sm, immunode®ciency, male (16,28±30). One interpretation of this conserved genetic infertility and a greatly elevated incidence of cancers of interaction is that RecQ helicases act upstream of topoisomer- most types (reviewed in 1). This predisposition to cancer is ase III in the same biochemical pathway and that RecQ thought to arise from the inherent genomic instability that is a helicases generate a DNA structure that requires resolution by feature of BS cells. In particular, BS cells display an elevated topoisomerase III (reviewed in 20). Consistent with this level of genetic recombination that is manifested as an proposal, E.coli RecQ can convert negatively supercoiled increase in the frequency of both sister chromatid exchanges plasmid DNA to a structure [as yet not de®ned, but presumed and interchromosomal homologous recombination events (2). to be single-stranded (ss)DNA] that can be acted upon by The gene mutated in BS, BLM, encodes a protein of molecular mass 159 kDa that belongs to the RecQ family of E.coli or S.cerevisiae Top3p to generate catenated DNA DNA helicases (3). BLM protein has been puri®ed and shown molecules (31). to act as a 3¢®5¢ DNA helicase on a variety of different DNA The S.cerevisiae Sgs1 and Top3 proteins also interact substrates (4±8). Mutations in two other genes encoding RecQ physically, raising the possibility that Sgs1p may recruit helicases are also associated with human cancer-prone Top3p to its site of action (16,32,33). We and others have disorders. WRN is defective in Werner's syndrome and demonstrated that BLM and human topoisomerase IIIa RECQ4 is defective in Rothmund±Thomson syndrome (hTOPO IIIa) are tightly associated in human cells (34±36) (9,10). Members of the RecQ helicase family contain a highly and that the two puri®ed proteins interact in vitro (35), conserved catalytic helicase domain that is ¯anked by domains indicating that this association is a direct one. that vary both in size and sequence between different family In this study, we demonstrate that BLM can stimulate the members. However, despite this apparent sequence diver- topoisomerase activity of hTOPO IIIa. In contrast, a mutant gence in those regions outside the helicase domain, all known BLM protein that is catalytically active, but no longer able to mutants lacking a RecQ helicase display genomic instability interact with hTOPO IIIa, has lost the ability to stimulate (reviewed in 11±13). Moreover, many of these mutants hTOPO IIIa protein. Moreover, we provide evidence that *To whom correspondence should be addressed. Tel: +44 1865 222 417; Fax: +44 1865 222 431; Email: ian.hickson@cancer.org.uk Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 4824 Nucleic Acids Research, 2002, Vol. 30 No. 22 hTOPO IIIa associates with a BLM±DNA complex. These products were performed using a PhosphorImager 840 data are consistent with the notion that hTOPO IIIa is (Molecular Dynamics) and ImageQuant software. recruited to its site of action through a direct interaction with Gel mobility shift assays the BLM helicase. Typically, BLM (300 nM) and hTOPO IIIa (100±900 nM) were incubated together with the labeled bubble-containing duplex substrate in 30 ml of reaction buffer (20 mM MATERIALS AND METHODS triethanolamine±HCl pH 7.5, 5 mM MgCl , 100 mg/ml BSA, DNA substrates 40 mM NaCl, 1 mM DTT and 5 mM ATPgS). Reactions were incubated at room temperature for 25 min. Protein±DNA The fX174 helicase substrate was generated by annealing a complexes were ®xed by the addition of 0.25% glutaraldehyde 21mer oligonucleotide (GTGCATATACCTGGTCTTTCG) and incubation at 37°C for 10 min, before electrophoresis to circular fX174 ssDNA before being extended by 4 nt in through a native 5% polyacrylamide gel in TBE buffer. the presence of Klenow polymerase, dATP, dTTP and [a- P]dCTP. G-quadruplex (G4) DNA representing the murine immunoglobulin Sg2B switch region and the 12 nt bubble-containing duplex were prepared using the oligo- RESULTS nucleotides and experimental conditions described previously BLM can stimulate the activity of hTOPO IIIa (6,7). Topoisomerase assays (see below) were performed on negatively supercoiled (form I) fX174 DNA. To examine a possible functional role for the interaction of the BLM and hTOPO IIIa proteins, we investigated whether BLM Expression and puri®cation of recombinant proteins had any effect on the ability of hTOPO IIIa to act upon Plasmids driving the expression of either hexahistidine-tagged negatively supercoiled fX174 DNA. When hTOPO IIIa was BLM or BLM-NC have been described previously (4,35). incubated with supercoiled fX174 DNA in the absence of Puri®cation of these proteins from yeast was as described by BLM, form I DNA disappeared with the concomitant appear- Karow et al. (4). Relative speci®c helicase activities of both ance of topoisomers. At longer incubation periods, fully proteins were determined using the partially double-stranded relaxed DNA (form II) was also evident. It is possible that a fX174 DNA substrate described above. Recombinant hTOPO proportion of form II DNA molecules also represented nicked IIIa was a kind gift of Drs Jean-Franc Ëois Riou and He Âle Áne DNA since Top3b from Drosophila melanogaster has been Goulaouic (Aventis Pharma, France). Human RPA was a kind shown to introduce single-stranded nicks into negatively gift of Dr Rick Wood (University of Pittsburgh). Escherichia supercoiled DNA (see Discussion). Given the potential coli SSB was purchased from Promega. heterogeneity of the reaction products generated by hTOPO IIIa, we quanti®ed the loss of form I DNA as an indication of Far-western analysis hTOPO IIIa activity and found that co-incubation with BLM Protein±protein interactions between hTOPO IIIa and the led to an approximate doubling in the rate of hTOPO IIIa BLM or BLM-NC proteins were tested as described previ- activity (Fig. 1A and B). Incubation of BLM alone with the ously (35). fX174 substrate had no effect on the level of form I DNA, indicating that the BLM preparation did not contain any Helicase assays contaminating topoisomerase activity (Fig. 1A). Unwinding of various DNA substrates by BLM and BLM-NC The stimulatory effect of BLM on the activity of hTOPO were performed using the reaction conditions described by IIIa was found to be dependent on the presence of RPA in the Karow et al. (4) reactions (Fig. 1C). It was therefore possible that RPA inhibits hTOPO IIIa by binding to ssDNA regions in the negatively Topoisomerase assays supercoiled substrate, thereby preventing access of hTOPO Typically, BLM (120 nM) and hTOPO IIIa (300 nM) were IIIa to the DNA. BLM might then act to stimulate hTOPO incubated with 200 ng of negatively supercoiled fX174 in the IIIa by displacing RPA from the DNA. To eliminate this presence of either human RPA (350 ng) or E.coli SSB (1.5 mg) possibility, we examined the effect of RPA on hTOPO IIIa in 30 ml of reaction buffer (50 mM Tris±HCl pH 7.5, 5 mM activity in the absence of BLM. RPA did not inhibit the MgCl , 100 mg/ml BSA, 40 mM NaCl, 0.2 U creatine kinase, plasmid relaxation activity of hTOPO IIIa, but rather had a 6 mM phosphocreatine and 1 mM DTT). In experiments mild stimulatory effect (Fig. 2). This effect appeared to be comparing BLM and BLM-NC, protein preparations were solely a function of RPA binding to ssDNA, as opposed to a diluted to give equivalent speci®c activities. Reactions were protein±protein interaction occurring between RPA and initiated by the addition of 5 mM ATP, followed by incubation hTOPO IIIa, since a similar stimulatory effect was also seen at 37°C. Aliquots of 5 ml were taken at the indicated times and when RPA was substituted by E.coli SSB (Fig. 2). We 1 mlof53 STOP buffer (250 mM EDTA, 5% SDS, 5 mg/ml therefore analysed whether SSB could substitute for RPA in proteinase K) was added. Samples were then incubated at supporting the stimulatory effects of BLM on the activity of 37°C for a further 10 min to deproteinise the DNA. The DNA hTOPO IIIa. Figure 2 shows that in the presence of SSB, BLM was separated on 0.6% agarose gels in the absence of ethidium still caused a stimulation of hTOPO IIIa plasmid relaxation bromide, before being transferred to nylon ®lters by conven- activity. Due to the apparent functional equivalence of RPA tional Southern blotting and then hybridised to a random- and SSB in these reactions, coupled with the commercial primed labeled fX174 DNA probe using Rediprime availability of SSB, the bacterial protein was used in all (Amersham). Visualisation and quanti®cation of reaction subsequent experiments. Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4825 Figure 1. BLM stimulates the activity of hTOPO IIIa.(A) Time course showing the relaxation of supercoiled fX174 DNA in the presence of 120 nM BLM alone (top), 300 nM hTOPO IIIa alone (middle) or BLM and hTOPO IIIa together (bottom). All reactions contained 150 nM RPA. The positions of supercoiled DNA (form I), relaxed DNA (form II) and intermediate topoisomers are indicated on the right. (B) Quanti®cation of the data from (A), showing loss of form I DNA in the presence of hTOPO IIIa alone (closed cicles) or BLM and hTOPO IIIa together (open circles). (C) Stimulation of hTOPO IIIa by BLM is dependent on RPA. Relaxation of supercoiled fX174 DNA incubated with various combinations of BLM, hTOPO IIIa and RPA, as indicated above the panel. Positions of supercoiled DNA (form I), relaxed DNA (form II) and topoisomers are indicated on the right. BLM can recruit hTOPO IIIa to single-stranded DNA negligible binding af®nity for the DNA substrate. However, bubbles the addition of hTOPO IIIa to BLM-containing reactions resulted in the conversion of 93% of B1 and 54% of B2 into a BLM and hTOPO IIIa have been shown to interact directly new, slower migrating complex, termed BT (Fig. 3). Since with each other and form a complex in vivo (34,35). Moreover, concentrations of hTOPO IIIa were used at which hTOPO it has been shown that the ability of ectopically expressed IIIa alone maximally bound <5% of the substrate, the BLM to reduce the elevated frequency of SCEs in BS cells conversion of the majority of B1 and B2 into BT indicates correlates with its ability to interact with hTOPO IIIa (36). that hTOPO IIIa preferentially binds B1 and B2 over the DNA The stimulatory effect of BLM on the activity of hTOPO IIIa substrate alone. These data also imply that DNA-bound BLM that we observed might therefore be mediated by the can still form a complex with hTOPO IIIa and are consistent recruitment of hTOPO IIIa to its site of action by BLM. In with the notion that BLM recruits hTOPO IIIa to its site of such a scenario, BLM should be able to simultaneously action on DNA. interact with both DNA and hTOPO IIIa. We have shown previously that BLM can unwind a duplex DNA molecule that Puri®cation of a hTOPO IIIa binding-defective form of contains a single-stranded bubble of the sort that is a BLM that retains helicase activity characteristic of negatively supercoiled DNA (6). We tested, therefore, the ability of BLM to bind simultaneously to a To con®rm that the stimulatory effect of BLM on hTOPO IIIa activity requires BLM to recruit hTOPO IIIa to its site of synthetic bubble-containing duplex DNA substrate and to hTOPO IIIa. As expected, BLM was found to bind the bubble- action, a mutant BLM protein was generated that was no containing substrate and generated two retarded complexes longer able to interact with hTOPO IIIa. Mapping studies designated B1 and B2 (Fig. 3). A proportion of the substrate have revealed that two hTOPO IIIa interaction domains exist was also incorporated into a complex that was retained in the in BLM that are located between residues 1±212 and 1267±1417 (35). A hexahistidine-tagged truncated protein, wells. Since this material did not resolve under the gel running conditions employed, it was not possible to anaylse further the BLM-NC, that consists of residues 213±1266 of BLM and nature of these apparent aggregates of DNA and protein. does not, therefore, contain either of the hTOPO IIIa Quanti®cation of the amount of DNA in these complexes interaction domains, was expressed in yeast and puri®ed to revealed that B1 and B2 represented 9 and 6%, respectively, of near homogeneity by nickel-chelate af®nity chromatography. BLM-NC had an apparent molecular mass of ~150 kDa on the total substrate in the reaction. In contrast, at the concentrations used in Figure 3, hTOPO IIIa displayed a SDS±PAGE (Fig. 4A) and was recognised on western blots by Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 4826 Nucleic Acids Research, 2002, Vol. 30 No. 22 Figure 2. Effects of RPA and SSB on plasmid relaxation catalysed by hTOPO IIIa.(A) Time course showing the relaxation of supercoiled fX174 DNA in the presence of combinations of BLM, hTOPO IIIIa, SSB or RPA, as indicated above the panels. Positions of supercoiled DNA (form I), relaxed DNA (form II) and topoisomers are indicated on the left. (B) Quanti®cation of the loss of form I DNA in the presence of hTOPO IIIa alone (open circles) or of hTOPO IIIa in the presence of RPA (open triangles) or SSB (open squares) or of hTOPO IIIa in the presence of BLM and SSB (closed squares). Figure 4. A truncated form of BLM that does not bind to hTOPO IIIa fails to stimulate topoisomerase activity. (A) A Coomassie blue stained poly- acrylamide gel of puri®ed BLM and BLM-NC (left) and a far-western blot (right) of the same BLM and BLM-NC proteins using hTOPO IIIa as probe (see text for details). (B) Comparison of the helicase activity of the BLM and BLM-NC proteins on substrates comprising an oligonucleotide annealed to single-stranded fX174 DNA (left) and G4 DNA (right). The positions of the substrate and the unwound ssDNA products are indicated on the left of each panel. Lanes marked ± contained no BLM protein. (C) Time course comparing the ability of BLM and BLM-NC to stimulate the topoisomerase activity of hTOPO IIIa on supercoiled fX174 DNA. Reactions contained hTOPO IIIa together with no additional protein (left), BLM-NC protein (middle) or full-length BLM protein (right). All reactions contained SSB. IIIa interaction domains might be present in the BLM protein not detected in our previous studies, far-western analysis using hTOPO IIIa as a probe was performed with BLM and BLM-NC. After separation of the BLM and BLM-NC proteins Figure 3. BLM can recruit hTOPO IIIa to ssDNA structures. by SDS±PAGE and transfer to nitrocellulose ®lters, the Electrophoretic mobility shift assay using a bubble DNA substrate, 300 nM membranes were incubated with hTOPO IIIa before being BLM (where indicated by + above the lanes) and varying concentrations of washed to remove any unbound material. hTOPO IIIa was hTOPO IIIa, as indicated above the lanes. The positions of the unbound DNA bubble substrate (end-labeled on one strand as indicated by the then detected by western analysis using a previously asterisk) and protein±DNA complexes (B1, B2 and BT) are indicated on the characterized polyclonal antibody (D6) (35). We have left. shown using this technique that hTOPO IIIa associates with full-length BLM (35), and this result was con®rmed in the current experiments (Fig. 4A). In contrast, hTOPO IIIa did not both polyclonal and monoclonal anti-BLM antibodies (35), as bind to BLM-NC (Fig. 4A), con®rming that all hTOPO IIIa well as by an anti-hexahistidine tag antibody (data not shown), interaction domains have been removed by truncation of BLM thereby con®rming its identity. to create BLM-NC. To establish that BLM-NC no longer bound hTOPO IIIa, Despite the fact that relatively large regions of BLM were and hence to eliminate the possibility that additional hTOPO deleted to generate BLM-NC, the truncated protein was still Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4827 catalytically active and was able to unwind a variety of DNA the expression of a fusion protein consisting of Top3p fused to substrates that have been shown to be substrates for the full- the N-terminus of a Top3p binding-defective form of Sgs1p length protein (4,6,7). These included oligonucleotides (32). Together, these data indicate that the evolutionarily annealed to a circular ssDNA and highly stable G4 DNA conserved interaction between RecQ helicases and topo- structures (Fig. 4B). isomerase III serves to recruit topoisomerase III to its site of action. A hTOPO IIIa binding-defective mutant form of BLM The requirement for the presence of either RPA or SSB in cannot stimulate hTOPO IIIa the reactions to observe the stimulatory effect of BLM on the We next compared the ability of BLM and BLM-NC to activity of hTOPO IIIa suggests that the DNA structure BLM stimulate the activity of hTOPO IIIa. Signi®cantly, the recruits hTOPO IIIa to has single-stranded character. stimulatory effect on hTOPO IIIa activity observed with Consistent with this is the ability of BLM to recruit hTOPO full-length BLM was not seen when BLM was substituted by IIIa to single-stranded `bubbles'. In human cells, the nature of BLM-NC (Fig. 4C). This failure of BLM-NC to stimulate the DNA structure that BLM loads hTOPO IIIa onto remains hTOPO IIIa was seen over a wide concentration range to be determined. TOPO IIIa is required for embryonic (81-fold), with higher concentrations even having a mild development in mice (42), indicating that TOPO IIIa performs inhibitory effect on hTOPO IIIa (Fig. 4C). We conclude, an essential role that cannot be provided by other topoisomer- therefore, that the stimulatory effect of BLM on hTOPO IIIa ases. Similarly, in both budding and ®ssion yeast, neither requires that the two proteins be capable of forming a Top1p nor Top2p can functionally substitute for Top3p complex. (26,27,29,30). Taken together, these ®ndings indicate that eukaryotic topoisomerase III enzymes do not function as typical topoisomerases and, consistent with this, it has been DISCUSSION reported previously that Top3p is unlikely to play a signi®cant In this paper, we report the ®rst demonstration of a functional role in regulating the overall supercoiling status of the budding biochemical interaction between a eukaryotic RecQ family yeast genome (reviewed in 43). Mutants lacking topoisomer- DNA helicase and topoisomerase III. BLM was found to ase III, as well as those defective in RecQ family helicases, signi®cantly stimulate the ability of hTOPO IIIa to act upon including BLM, generally display hyper-recombination negatively supercoiled DNA. When hTOPO IIIa alone was throughout the genome (14±19,26). This would suggest that incubated with supercoiled fX174 DNA, two classes of the BLM±hTOPO IIIa complex acts to suppress inadvertant reaction products were observed. These were in the form of recombination or to disrupt inappropriately paired DNA topoisomers that appeared after 15 min incubation, and form II molecules. One possible target for the complex is the DNA that only accumulated after longer periods of incubation, Holliday junction recombination intermediate. It is known up to 60 min. The latter class of reaction products most likely that RecQ, Sgs1p, WRN and BLM can disrupt Holliday consisted of fully relaxed DNA, since their appearance junctions (5,6,44± 46). Moreover, we have shown recently that occurred only after the formation of topoisomers. However, BLM promotes the ATP-dependent branch migration of these it is also possible that a proportion of form II molecules junctions (5). Through catalysing this reaction, BLM may act to promote and/or eliminate recombinants, depending upon contained single strand nicks. Indeed, Wilson-Sali and Hsieh the circumstances. Although the role of topoisomerase III in (37) have reported recently that Top3b from D.melanogaster this process is unclear, it may be signi®cant that yeast Top3p is able to catalyse the nicking of negatively supercoiled DNA. has been shown to be required for the resolution of meiotic It is presently unknown if hTOPO IIIa possesses an equivalent recombination intermediates (27). The possibility exists, endonucleolytic activity. However, we are currently address- therefore, that BLM recruits hTOPO IIIa to Holliday ing this issue and determining what differential effects BLM junctions to affect their resolution. Ongoing studies of the might have on the topoisomerase versus putative endonu- effects of hTOPO IIIa on BLM-catalysed Holliday junction clease activities of hTOPO IIIa. branch migration reactions aim to address this possibility. A BLM was found able to bind simultaneously to both hTOPO IIIa and DNA. Moreover, the stimulation of hTOPO IIIa by second potential role for the BLM±hTOPO IIIa complex is in BLM was lost when BLM was modi®ed to eliminate all of the the elimination of G-quadruplex DNA in order to permit hTOPO IIIa interaction domains. We therefore propose that progression of the replication and/or transcription machinery. one role of BLM is to recruit hTOPO IIIa to its site of action. This ability of BLM to unwind such non-canonical This proposal is supported by a number of observations. In Watson±Crick DNA structures is a conserved function of the normal cells, BLM and hTOPO IIIa can be detected together RecQ family helicases (13). G4 DNA has been suggested to be in subnuclear structures termed PML bodies (38±41). highly recombinogenic due primarily to its potential to lead to However, in BS cells, hTOPO IIIa is expressed normally replication fork stalling and hence the formation of DNA but is aberrantly localised in the nucleus (34,35). Furthermore, double-strand breaks. recent studies on Sgs1p, the budding yeast homologue of In summary, we have shown that BLM stimulates the BLM, which also interacts with Top3p, have shown that activity of hTOPO IIIa and that this stimulation requires that expression of mutant forms of Sgs1p that cannot associate the two proteins be able to form a stable complex. We propose withTop3p are unable to complement several aspects of the that BLM functions to regulate the levels of genetic sgs1 phenotype, including sensitivity to methylmethane recombination through the recruitment of hTOPO IIIa to sulphonate and hydroxyurea, which damage DNA and inhibit recombinogenic DNA structures and/or recombination inter- DNA replication, respectively (32). However, this require- mediates. The biochemical functions of BLM and hTOPO IIIa ment for Sgs1p to interact with Top3p can be circumvented by appear to be intimately connected, consistent with the Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 4828 Nucleic Acids Research, 2002, Vol. 30 No. 22 18. Watt,P.M., Hickson,I.D., Borts,R.H. and Louis,E.J. (1996) SGS1, a observation that lack of BLM in BS cell lines causes hTOPO homologue of the Bloom's and Werner's syndrome genes, is required for IIIa to be mislocalised in the nucleus (34,35). It is therefore maintenance of genome stability in Saccharomyces cerevisiae. Genetics, quite possible that the diverse phenotypes observed in BS cells 144, 935±945. are not due solely to a loss of BLM. Instead, `uncoupling' of 19. Nakayama,H., Nakayama,K., Nakayama,R., Irino,N., Nakayama,Y. and Hanawalt,P.C. (1984) Isolation and genetic characterization of a the BLM±hTOPO IIIa heteromeric helicase/topoisomerase thymineless death-resistant mutant of Escherichia coli K12: complex might be at least partially responsible for this identi®cation of a new mutation (recQ1) that blocks the RecF phenotypic diversity. recombination pathway. Mol. Gen. Genet., 195, 474±480. 20. Wu,L. and Hickson,I.D. (2001) RecQ helicases and topoisomerases: components of a conserved complex for the regulation of genetic recombination. Cell. Mol. Life Sci., 58, 894±901. ACKNOWLEDGEMENTS 21. Champoux,J.J. (2001) DNA topoisomerases: structure, function and We thank Drs J.-F. Riou and H. Goulaouic for hTOPO IIIa, mechanism. Annu. Rev. Biochem., 70, 369±413. 22. Hanai,R., Caron,P.R. and Wang,J.C. (1996) Human TOP3: a single-copy Dr R. Wood for RPA, Dr C. Norbury for critical reading of the gene encoding DNA topoisomerase III. Proc. Natl Acad. Sci. USA, 93, manuscript and members of the Cancer Research UK Genome 3653±3657. Integrity Group for useful discussions. This work was 23. Seki,T., Seki,M., Onodera,R., Katada,T. and Enomoto,T. (1998) Cloning supported by Cancer Research UK. of cDNA encoding a novel mouse DNA topoisomerase III (Topo IIIbeta) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis. J. Biol. Chem., 273, 28553±28556. 24. 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Bennett,R.J., Keck,J.L. and Wang,J.C. (1999) Binding speci®city (2001) Regulation and localization of the Bloom syndrome protein in determines polarity of DNA unwinding by the Sgs1 protein of S. response to DNA damage. J. Cell Biol., 153, 367±380. cerevisiae. J. Mol. Biol., 289, 235±248. 41. Ishov,A.M., Sotnikov,A.G., Negorev,D., Vladimirova,O.V., Neff,N., 45. Constantinou,A., Tarsounas,M., Karow,J.K., Brosh,R.M., Bohr,V.A., Kamitani,T., Yeh,E.T., Strauss,J.F.,III and Maul,G.G. (1999) PML is Hickson,I.D. and West,S.C. (2000) Werner's syndrome protein (WRN) critical for ND10 formation and recruits the PML-interacting protein migrates Holliday junctions and co-localises with RPA upon replication daxx to this nuclear structure when modi®ed by SUMO-1. J. Cell Biol., arrest. EMBO Rep., 1, 80±84. 147, 221±234. 46. Harmon,F.G. and Kowalczykowski,S.C. (1998) RecQ helicase, in 42. Li,W. and Wang,J.C. (1998) Mammalian DNA topoisomerase IIIalpha is concert with RecA and SSB proteins, initiates and disrupts DNA essential in early embryogenesis. Proc. Natl Acad. Sci. USA, 95, recombination. Genes Dev., 12, 1134±1144. 1010±1013. Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

The Bloom’s syndrome helicase stimulates the activity of human topoisomerase IIIα

Nucleic Acids Research , Volume 30 (22) – Nov 15, 2002

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10.1093/nar/gkf611
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Abstract

ã 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30 No. 22 4823±4829 The Bloom's syndrome helicase stimulates the activity of human topoisomerase IIIa Leonard Wu and Ian D. Hickson* Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK Received August 15, 2002; Revised and Accepted September 17, 2002 ABSTRACT display a hyper-recombinogenic phenotype reminiscent of BS cells, suggesting that RecQ helicases perform a conserved Bloom's syndrome (BS) is a disorder associated function in controlling the level of homologous recombination with chromosomal instability and a predisposition in cells (14±19). Although the precise cellular role of any to the development of cancer. The BS gene product, RecQ helicase has yet to be elucidated, several lines of BLM, is a DNA helicase of the RecQ family that forms evidence suggest that RecQ helicases act in concert with type a complex in vitro and in vivo with topoisomerase IA topoisomerases (reviewed in 20). IIIa. Here, we show that BLM stimulates the ability of The type IA subclass of topoisomerases includes Escherichia coli topoisomerases I and III and the eukaryotic topoisomerase IIIa to relax negatively supercoiled topoisomerase III enzymes (reviewed in 21). In vertebrates, DNA. Moreover, DNA binding analyses indicate that there are at least two isoforms of topoisomerase III, termed a BLM recruits topoisomerase IIIa to its DNA sub- and b, which display only a weak topoisomerase activity strate. Consistent with this, a mutant form of BLM towards negatively supercoiled DNA (22±25). Yeast cells that retains helicase activity, but is unable to bind express a single topoisomerase III enzyme encoded by the topoisomerase IIIa, fails to stimulate topoisomerase TOP3 gene (26). In Saccharomyces cerevisiae, top3D mutants activity. These results indicate that a physical are viable, but grow very slowly and have defects in S phase association between BLM and topoisomerase IIIa responses to DNA damage and in both mitotic and meiotic is a prerequisite for their functional biochemical recombination (16,26±28). In contrast, the top3 gene in interaction. Schizosaccharomyces pombe is essential for viability, with top3D mutants displaying an inability to accurately segregate daughter chromosomes during mitosis (29,30). Interestingly, INTRODUCTION mutation of SGS1 or rqh1 , the sole RecQ homologues found in budding and ®ssion yeast, respectively, can suppress the Bloom's syndrome (BS) is a rare genetic disorder character- deleterious effects caused by the absence of Top3 protein ized by proportional dwar®sm, immunode®ciency, male (16,28±30). One interpretation of this conserved genetic infertility and a greatly elevated incidence of cancers of interaction is that RecQ helicases act upstream of topoisomer- most types (reviewed in 1). This predisposition to cancer is ase III in the same biochemical pathway and that RecQ thought to arise from the inherent genomic instability that is a helicases generate a DNA structure that requires resolution by feature of BS cells. In particular, BS cells display an elevated topoisomerase III (reviewed in 20). Consistent with this level of genetic recombination that is manifested as an proposal, E.coli RecQ can convert negatively supercoiled increase in the frequency of both sister chromatid exchanges plasmid DNA to a structure [as yet not de®ned, but presumed and interchromosomal homologous recombination events (2). to be single-stranded (ss)DNA] that can be acted upon by The gene mutated in BS, BLM, encodes a protein of molecular mass 159 kDa that belongs to the RecQ family of E.coli or S.cerevisiae Top3p to generate catenated DNA DNA helicases (3). BLM protein has been puri®ed and shown molecules (31). to act as a 3¢®5¢ DNA helicase on a variety of different DNA The S.cerevisiae Sgs1 and Top3 proteins also interact substrates (4±8). Mutations in two other genes encoding RecQ physically, raising the possibility that Sgs1p may recruit helicases are also associated with human cancer-prone Top3p to its site of action (16,32,33). We and others have disorders. WRN is defective in Werner's syndrome and demonstrated that BLM and human topoisomerase IIIa RECQ4 is defective in Rothmund±Thomson syndrome (hTOPO IIIa) are tightly associated in human cells (34±36) (9,10). Members of the RecQ helicase family contain a highly and that the two puri®ed proteins interact in vitro (35), conserved catalytic helicase domain that is ¯anked by domains indicating that this association is a direct one. that vary both in size and sequence between different family In this study, we demonstrate that BLM can stimulate the members. However, despite this apparent sequence diver- topoisomerase activity of hTOPO IIIa. In contrast, a mutant gence in those regions outside the helicase domain, all known BLM protein that is catalytically active, but no longer able to mutants lacking a RecQ helicase display genomic instability interact with hTOPO IIIa, has lost the ability to stimulate (reviewed in 11±13). Moreover, many of these mutants hTOPO IIIa protein. Moreover, we provide evidence that *To whom correspondence should be addressed. Tel: +44 1865 222 417; Fax: +44 1865 222 431; Email: ian.hickson@cancer.org.uk Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 4824 Nucleic Acids Research, 2002, Vol. 30 No. 22 hTOPO IIIa associates with a BLM±DNA complex. These products were performed using a PhosphorImager 840 data are consistent with the notion that hTOPO IIIa is (Molecular Dynamics) and ImageQuant software. recruited to its site of action through a direct interaction with Gel mobility shift assays the BLM helicase. Typically, BLM (300 nM) and hTOPO IIIa (100±900 nM) were incubated together with the labeled bubble-containing duplex substrate in 30 ml of reaction buffer (20 mM MATERIALS AND METHODS triethanolamine±HCl pH 7.5, 5 mM MgCl , 100 mg/ml BSA, DNA substrates 40 mM NaCl, 1 mM DTT and 5 mM ATPgS). Reactions were incubated at room temperature for 25 min. Protein±DNA The fX174 helicase substrate was generated by annealing a complexes were ®xed by the addition of 0.25% glutaraldehyde 21mer oligonucleotide (GTGCATATACCTGGTCTTTCG) and incubation at 37°C for 10 min, before electrophoresis to circular fX174 ssDNA before being extended by 4 nt in through a native 5% polyacrylamide gel in TBE buffer. the presence of Klenow polymerase, dATP, dTTP and [a- P]dCTP. G-quadruplex (G4) DNA representing the murine immunoglobulin Sg2B switch region and the 12 nt bubble-containing duplex were prepared using the oligo- RESULTS nucleotides and experimental conditions described previously BLM can stimulate the activity of hTOPO IIIa (6,7). Topoisomerase assays (see below) were performed on negatively supercoiled (form I) fX174 DNA. To examine a possible functional role for the interaction of the BLM and hTOPO IIIa proteins, we investigated whether BLM Expression and puri®cation of recombinant proteins had any effect on the ability of hTOPO IIIa to act upon Plasmids driving the expression of either hexahistidine-tagged negatively supercoiled fX174 DNA. When hTOPO IIIa was BLM or BLM-NC have been described previously (4,35). incubated with supercoiled fX174 DNA in the absence of Puri®cation of these proteins from yeast was as described by BLM, form I DNA disappeared with the concomitant appear- Karow et al. (4). Relative speci®c helicase activities of both ance of topoisomers. At longer incubation periods, fully proteins were determined using the partially double-stranded relaxed DNA (form II) was also evident. It is possible that a fX174 DNA substrate described above. Recombinant hTOPO proportion of form II DNA molecules also represented nicked IIIa was a kind gift of Drs Jean-Franc Ëois Riou and He Âle Áne DNA since Top3b from Drosophila melanogaster has been Goulaouic (Aventis Pharma, France). Human RPA was a kind shown to introduce single-stranded nicks into negatively gift of Dr Rick Wood (University of Pittsburgh). Escherichia supercoiled DNA (see Discussion). Given the potential coli SSB was purchased from Promega. heterogeneity of the reaction products generated by hTOPO IIIa, we quanti®ed the loss of form I DNA as an indication of Far-western analysis hTOPO IIIa activity and found that co-incubation with BLM Protein±protein interactions between hTOPO IIIa and the led to an approximate doubling in the rate of hTOPO IIIa BLM or BLM-NC proteins were tested as described previ- activity (Fig. 1A and B). Incubation of BLM alone with the ously (35). fX174 substrate had no effect on the level of form I DNA, indicating that the BLM preparation did not contain any Helicase assays contaminating topoisomerase activity (Fig. 1A). Unwinding of various DNA substrates by BLM and BLM-NC The stimulatory effect of BLM on the activity of hTOPO were performed using the reaction conditions described by IIIa was found to be dependent on the presence of RPA in the Karow et al. (4) reactions (Fig. 1C). It was therefore possible that RPA inhibits hTOPO IIIa by binding to ssDNA regions in the negatively Topoisomerase assays supercoiled substrate, thereby preventing access of hTOPO Typically, BLM (120 nM) and hTOPO IIIa (300 nM) were IIIa to the DNA. BLM might then act to stimulate hTOPO incubated with 200 ng of negatively supercoiled fX174 in the IIIa by displacing RPA from the DNA. To eliminate this presence of either human RPA (350 ng) or E.coli SSB (1.5 mg) possibility, we examined the effect of RPA on hTOPO IIIa in 30 ml of reaction buffer (50 mM Tris±HCl pH 7.5, 5 mM activity in the absence of BLM. RPA did not inhibit the MgCl , 100 mg/ml BSA, 40 mM NaCl, 0.2 U creatine kinase, plasmid relaxation activity of hTOPO IIIa, but rather had a 6 mM phosphocreatine and 1 mM DTT). In experiments mild stimulatory effect (Fig. 2). This effect appeared to be comparing BLM and BLM-NC, protein preparations were solely a function of RPA binding to ssDNA, as opposed to a diluted to give equivalent speci®c activities. Reactions were protein±protein interaction occurring between RPA and initiated by the addition of 5 mM ATP, followed by incubation hTOPO IIIa, since a similar stimulatory effect was also seen at 37°C. Aliquots of 5 ml were taken at the indicated times and when RPA was substituted by E.coli SSB (Fig. 2). We 1 mlof53 STOP buffer (250 mM EDTA, 5% SDS, 5 mg/ml therefore analysed whether SSB could substitute for RPA in proteinase K) was added. Samples were then incubated at supporting the stimulatory effects of BLM on the activity of 37°C for a further 10 min to deproteinise the DNA. The DNA hTOPO IIIa. Figure 2 shows that in the presence of SSB, BLM was separated on 0.6% agarose gels in the absence of ethidium still caused a stimulation of hTOPO IIIa plasmid relaxation bromide, before being transferred to nylon ®lters by conven- activity. Due to the apparent functional equivalence of RPA tional Southern blotting and then hybridised to a random- and SSB in these reactions, coupled with the commercial primed labeled fX174 DNA probe using Rediprime availability of SSB, the bacterial protein was used in all (Amersham). Visualisation and quanti®cation of reaction subsequent experiments. Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4825 Figure 1. BLM stimulates the activity of hTOPO IIIa.(A) Time course showing the relaxation of supercoiled fX174 DNA in the presence of 120 nM BLM alone (top), 300 nM hTOPO IIIa alone (middle) or BLM and hTOPO IIIa together (bottom). All reactions contained 150 nM RPA. The positions of supercoiled DNA (form I), relaxed DNA (form II) and intermediate topoisomers are indicated on the right. (B) Quanti®cation of the data from (A), showing loss of form I DNA in the presence of hTOPO IIIa alone (closed cicles) or BLM and hTOPO IIIa together (open circles). (C) Stimulation of hTOPO IIIa by BLM is dependent on RPA. Relaxation of supercoiled fX174 DNA incubated with various combinations of BLM, hTOPO IIIa and RPA, as indicated above the panel. Positions of supercoiled DNA (form I), relaxed DNA (form II) and topoisomers are indicated on the right. BLM can recruit hTOPO IIIa to single-stranded DNA negligible binding af®nity for the DNA substrate. However, bubbles the addition of hTOPO IIIa to BLM-containing reactions resulted in the conversion of 93% of B1 and 54% of B2 into a BLM and hTOPO IIIa have been shown to interact directly new, slower migrating complex, termed BT (Fig. 3). Since with each other and form a complex in vivo (34,35). Moreover, concentrations of hTOPO IIIa were used at which hTOPO it has been shown that the ability of ectopically expressed IIIa alone maximally bound <5% of the substrate, the BLM to reduce the elevated frequency of SCEs in BS cells conversion of the majority of B1 and B2 into BT indicates correlates with its ability to interact with hTOPO IIIa (36). that hTOPO IIIa preferentially binds B1 and B2 over the DNA The stimulatory effect of BLM on the activity of hTOPO IIIa substrate alone. These data also imply that DNA-bound BLM that we observed might therefore be mediated by the can still form a complex with hTOPO IIIa and are consistent recruitment of hTOPO IIIa to its site of action by BLM. In with the notion that BLM recruits hTOPO IIIa to its site of such a scenario, BLM should be able to simultaneously action on DNA. interact with both DNA and hTOPO IIIa. We have shown previously that BLM can unwind a duplex DNA molecule that Puri®cation of a hTOPO IIIa binding-defective form of contains a single-stranded bubble of the sort that is a BLM that retains helicase activity characteristic of negatively supercoiled DNA (6). We tested, therefore, the ability of BLM to bind simultaneously to a To con®rm that the stimulatory effect of BLM on hTOPO IIIa activity requires BLM to recruit hTOPO IIIa to its site of synthetic bubble-containing duplex DNA substrate and to hTOPO IIIa. As expected, BLM was found to bind the bubble- action, a mutant BLM protein was generated that was no containing substrate and generated two retarded complexes longer able to interact with hTOPO IIIa. Mapping studies designated B1 and B2 (Fig. 3). A proportion of the substrate have revealed that two hTOPO IIIa interaction domains exist was also incorporated into a complex that was retained in the in BLM that are located between residues 1±212 and 1267±1417 (35). A hexahistidine-tagged truncated protein, wells. Since this material did not resolve under the gel running conditions employed, it was not possible to anaylse further the BLM-NC, that consists of residues 213±1266 of BLM and nature of these apparent aggregates of DNA and protein. does not, therefore, contain either of the hTOPO IIIa Quanti®cation of the amount of DNA in these complexes interaction domains, was expressed in yeast and puri®ed to revealed that B1 and B2 represented 9 and 6%, respectively, of near homogeneity by nickel-chelate af®nity chromatography. BLM-NC had an apparent molecular mass of ~150 kDa on the total substrate in the reaction. In contrast, at the concentrations used in Figure 3, hTOPO IIIa displayed a SDS±PAGE (Fig. 4A) and was recognised on western blots by Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 4826 Nucleic Acids Research, 2002, Vol. 30 No. 22 Figure 2. Effects of RPA and SSB on plasmid relaxation catalysed by hTOPO IIIa.(A) Time course showing the relaxation of supercoiled fX174 DNA in the presence of combinations of BLM, hTOPO IIIIa, SSB or RPA, as indicated above the panels. Positions of supercoiled DNA (form I), relaxed DNA (form II) and topoisomers are indicated on the left. (B) Quanti®cation of the loss of form I DNA in the presence of hTOPO IIIa alone (open circles) or of hTOPO IIIa in the presence of RPA (open triangles) or SSB (open squares) or of hTOPO IIIa in the presence of BLM and SSB (closed squares). Figure 4. A truncated form of BLM that does not bind to hTOPO IIIa fails to stimulate topoisomerase activity. (A) A Coomassie blue stained poly- acrylamide gel of puri®ed BLM and BLM-NC (left) and a far-western blot (right) of the same BLM and BLM-NC proteins using hTOPO IIIa as probe (see text for details). (B) Comparison of the helicase activity of the BLM and BLM-NC proteins on substrates comprising an oligonucleotide annealed to single-stranded fX174 DNA (left) and G4 DNA (right). The positions of the substrate and the unwound ssDNA products are indicated on the left of each panel. Lanes marked ± contained no BLM protein. (C) Time course comparing the ability of BLM and BLM-NC to stimulate the topoisomerase activity of hTOPO IIIa on supercoiled fX174 DNA. Reactions contained hTOPO IIIa together with no additional protein (left), BLM-NC protein (middle) or full-length BLM protein (right). All reactions contained SSB. IIIa interaction domains might be present in the BLM protein not detected in our previous studies, far-western analysis using hTOPO IIIa as a probe was performed with BLM and BLM-NC. After separation of the BLM and BLM-NC proteins Figure 3. BLM can recruit hTOPO IIIa to ssDNA structures. by SDS±PAGE and transfer to nitrocellulose ®lters, the Electrophoretic mobility shift assay using a bubble DNA substrate, 300 nM membranes were incubated with hTOPO IIIa before being BLM (where indicated by + above the lanes) and varying concentrations of washed to remove any unbound material. hTOPO IIIa was hTOPO IIIa, as indicated above the lanes. The positions of the unbound DNA bubble substrate (end-labeled on one strand as indicated by the then detected by western analysis using a previously asterisk) and protein±DNA complexes (B1, B2 and BT) are indicated on the characterized polyclonal antibody (D6) (35). We have left. shown using this technique that hTOPO IIIa associates with full-length BLM (35), and this result was con®rmed in the current experiments (Fig. 4A). In contrast, hTOPO IIIa did not both polyclonal and monoclonal anti-BLM antibodies (35), as bind to BLM-NC (Fig. 4A), con®rming that all hTOPO IIIa well as by an anti-hexahistidine tag antibody (data not shown), interaction domains have been removed by truncation of BLM thereby con®rming its identity. to create BLM-NC. To establish that BLM-NC no longer bound hTOPO IIIa, Despite the fact that relatively large regions of BLM were and hence to eliminate the possibility that additional hTOPO deleted to generate BLM-NC, the truncated protein was still Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4827 catalytically active and was able to unwind a variety of DNA the expression of a fusion protein consisting of Top3p fused to substrates that have been shown to be substrates for the full- the N-terminus of a Top3p binding-defective form of Sgs1p length protein (4,6,7). These included oligonucleotides (32). Together, these data indicate that the evolutionarily annealed to a circular ssDNA and highly stable G4 DNA conserved interaction between RecQ helicases and topo- structures (Fig. 4B). isomerase III serves to recruit topoisomerase III to its site of action. A hTOPO IIIa binding-defective mutant form of BLM The requirement for the presence of either RPA or SSB in cannot stimulate hTOPO IIIa the reactions to observe the stimulatory effect of BLM on the We next compared the ability of BLM and BLM-NC to activity of hTOPO IIIa suggests that the DNA structure BLM stimulate the activity of hTOPO IIIa. Signi®cantly, the recruits hTOPO IIIa to has single-stranded character. stimulatory effect on hTOPO IIIa activity observed with Consistent with this is the ability of BLM to recruit hTOPO full-length BLM was not seen when BLM was substituted by IIIa to single-stranded `bubbles'. In human cells, the nature of BLM-NC (Fig. 4C). This failure of BLM-NC to stimulate the DNA structure that BLM loads hTOPO IIIa onto remains hTOPO IIIa was seen over a wide concentration range to be determined. TOPO IIIa is required for embryonic (81-fold), with higher concentrations even having a mild development in mice (42), indicating that TOPO IIIa performs inhibitory effect on hTOPO IIIa (Fig. 4C). We conclude, an essential role that cannot be provided by other topoisomer- therefore, that the stimulatory effect of BLM on hTOPO IIIa ases. Similarly, in both budding and ®ssion yeast, neither requires that the two proteins be capable of forming a Top1p nor Top2p can functionally substitute for Top3p complex. (26,27,29,30). Taken together, these ®ndings indicate that eukaryotic topoisomerase III enzymes do not function as typical topoisomerases and, consistent with this, it has been DISCUSSION reported previously that Top3p is unlikely to play a signi®cant In this paper, we report the ®rst demonstration of a functional role in regulating the overall supercoiling status of the budding biochemical interaction between a eukaryotic RecQ family yeast genome (reviewed in 43). Mutants lacking topoisomer- DNA helicase and topoisomerase III. BLM was found to ase III, as well as those defective in RecQ family helicases, signi®cantly stimulate the ability of hTOPO IIIa to act upon including BLM, generally display hyper-recombination negatively supercoiled DNA. When hTOPO IIIa alone was throughout the genome (14±19,26). This would suggest that incubated with supercoiled fX174 DNA, two classes of the BLM±hTOPO IIIa complex acts to suppress inadvertant reaction products were observed. These were in the form of recombination or to disrupt inappropriately paired DNA topoisomers that appeared after 15 min incubation, and form II molecules. One possible target for the complex is the DNA that only accumulated after longer periods of incubation, Holliday junction recombination intermediate. It is known up to 60 min. The latter class of reaction products most likely that RecQ, Sgs1p, WRN and BLM can disrupt Holliday consisted of fully relaxed DNA, since their appearance junctions (5,6,44± 46). Moreover, we have shown recently that occurred only after the formation of topoisomers. However, BLM promotes the ATP-dependent branch migration of these it is also possible that a proportion of form II molecules junctions (5). Through catalysing this reaction, BLM may act to promote and/or eliminate recombinants, depending upon contained single strand nicks. Indeed, Wilson-Sali and Hsieh the circumstances. Although the role of topoisomerase III in (37) have reported recently that Top3b from D.melanogaster this process is unclear, it may be signi®cant that yeast Top3p is able to catalyse the nicking of negatively supercoiled DNA. has been shown to be required for the resolution of meiotic It is presently unknown if hTOPO IIIa possesses an equivalent recombination intermediates (27). The possibility exists, endonucleolytic activity. However, we are currently address- therefore, that BLM recruits hTOPO IIIa to Holliday ing this issue and determining what differential effects BLM junctions to affect their resolution. Ongoing studies of the might have on the topoisomerase versus putative endonu- effects of hTOPO IIIa on BLM-catalysed Holliday junction clease activities of hTOPO IIIa. branch migration reactions aim to address this possibility. A BLM was found able to bind simultaneously to both hTOPO IIIa and DNA. Moreover, the stimulation of hTOPO IIIa by second potential role for the BLM±hTOPO IIIa complex is in BLM was lost when BLM was modi®ed to eliminate all of the the elimination of G-quadruplex DNA in order to permit hTOPO IIIa interaction domains. We therefore propose that progression of the replication and/or transcription machinery. one role of BLM is to recruit hTOPO IIIa to its site of action. This ability of BLM to unwind such non-canonical This proposal is supported by a number of observations. In Watson±Crick DNA structures is a conserved function of the normal cells, BLM and hTOPO IIIa can be detected together RecQ family helicases (13). G4 DNA has been suggested to be in subnuclear structures termed PML bodies (38±41). highly recombinogenic due primarily to its potential to lead to However, in BS cells, hTOPO IIIa is expressed normally replication fork stalling and hence the formation of DNA but is aberrantly localised in the nucleus (34,35). Furthermore, double-strand breaks. recent studies on Sgs1p, the budding yeast homologue of In summary, we have shown that BLM stimulates the BLM, which also interacts with Top3p, have shown that activity of hTOPO IIIa and that this stimulation requires that expression of mutant forms of Sgs1p that cannot associate the two proteins be able to form a stable complex. We propose withTop3p are unable to complement several aspects of the that BLM functions to regulate the levels of genetic sgs1 phenotype, including sensitivity to methylmethane recombination through the recruitment of hTOPO IIIa to sulphonate and hydroxyurea, which damage DNA and inhibit recombinogenic DNA structures and/or recombination inter- DNA replication, respectively (32). However, this require- mediates. The biochemical functions of BLM and hTOPO IIIa ment for Sgs1p to interact with Top3p can be circumvented by appear to be intimately connected, consistent with the Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4823/2380441 by Ed 'DeepDyve' Gillespie user on 06 February 2018 4828 Nucleic Acids Research, 2002, Vol. 30 No. 22 18. Watt,P.M., Hickson,I.D., Borts,R.H. and Louis,E.J. (1996) SGS1, a observation that lack of BLM in BS cell lines causes hTOPO homologue of the Bloom's and Werner's syndrome genes, is required for IIIa to be mislocalised in the nucleus (34,35). It is therefore maintenance of genome stability in Saccharomyces cerevisiae. Genetics, quite possible that the diverse phenotypes observed in BS cells 144, 935±945. are not due solely to a loss of BLM. Instead, `uncoupling' of 19. Nakayama,H., Nakayama,K., Nakayama,R., Irino,N., Nakayama,Y. and Hanawalt,P.C. (1984) Isolation and genetic characterization of a the BLM±hTOPO IIIa heteromeric helicase/topoisomerase thymineless death-resistant mutant of Escherichia coli K12: complex might be at least partially responsible for this identi®cation of a new mutation (recQ1) that blocks the RecF phenotypic diversity. recombination pathway. Mol. Gen. Genet., 195, 474±480. 20. Wu,L. and Hickson,I.D. 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Nucleic Acids ResearchOxford University Press

Published: Nov 15, 2002

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