The Bloom's Syndrome Helicase Can Promote the Regression of a Model Replication Fork

Abstract

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called “chicken foot” structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges. Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called “chicken foot” structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges. In every cell cycle, the replication apparatus encounters numerous chemical and protein blocks that can perturb fork progression (1Rothstein R. Michel B. Gangloff S. Genes Dev. 2000; 14: 1-10Crossref PubMed Google Scholar). The consequences of such encounters and the subsequent replication re-start processes employed will depend on both the type of lesion and on the template strand on which the damage resides. Lesions on the lagging strand template can, theoretically, be by-passed via the priming of an Okazaki fragment downstream of the lesion resulting in a gap in the nascent lagging strand. These gaps can then be filled, post-replicatively, by error-prone translesion polymerases or repaired in an error-free manner by homologous recombination (1Rothstein R. Michel B. Gangloff S. Genes Dev. 2000; 14: 1-10Crossref PubMed Google Scholar, 2Friedberg E.C. Nat. Rev. Mol. Cell Biol. 2005; 6: 943-953Crossref PubMed Scopus (211) Google Scholar). Lesions on the leading strand template, however, may cause the replisome to stall. On such occasions, lagging strand synthesis can become uncoupled from leading strand synthesis, with the result that the lagging strand is extended beyond the site of the lesion (3Higuchi K. Katayama T. Iwai S. Hidaka M. Horiuchi T. Maki H. Genes Cells. 2003; 8: 437-449Crossref PubMed Scopus (122) Google Scholar, 4McInerney P. O'Donnell M. J. Biol. Chem. 2004; 279: 21543-21551Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 5Pages V. Fuchs R.P. Science. 2003; 300: 1300-1303Crossref PubMed Scopus (176) Google Scholar, 6Svoboda D.L. Vos J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11975-11979Crossref PubMed Scopus (100) Google Scholar). This apparently futile extension of the nascent lagging strand may actually serve an important role in lesion bypass. If, as proposed, the fork were to regress via annealing of the nascent strands, the longer lagging strand can provide a template for the prematurely terminated leading strand to be extended. In this way, by a process of template switching, leading strand synthesis can be continued beyond the point of the block on the parental template, effectively “bypassing” the lesion. The fork can then be re-established to allow leading strand synthesis to re-commence through either reversal of the regressed fork or recombination-dependent invasion of the regressed arm into the parental template at a point downstream of the lesion (7Cox M.M. Annu. Rev. Genet. 2001; 35: 53-82Crossref PubMed Scopus (199) Google Scholar, 8McGlynn P. Lloyd R.G. Trends Genet. 2002; 18: 413-419Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In Escherichia coli, a number of proteins have been shown to promote the regression of stalled forks, which include the DNA helicase RecG and the recombinase protein RecA (9McGlynn P. Lloyd R.G. Cell. 2000; 101: 35-45Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 10Robu M.E. Inman R.B. Cox M.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8211-8218Crossref PubMed Scopus (128) Google Scholar). These proteins are unrelated functionally, which suggests that multiple mechanisms may exist to promote fork regression that could be utilized under different circumstances of replication fork repair (11Robu M.E. Inman R.B. Cox M.M. J. Biol. Chem. 2004; 279: 10973-10981Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In eukaryotes, the mechanisms for processing stalled forks is less well characterized. Nevertheless, several lines of evidence indicate that fork regression might occur at eukaryotic replication forks (12Sogo J.M. Lopes M. Foiani M. Science. 2002; 297: 599-602Crossref PubMed Scopus (678) Google Scholar, 13Zou H. Rothstein R. Cell. 1997; 90: 87-96Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). However, how the process of fork regression is facilitated in eukaryotes is, as yet, unclear. The Bloom's syndrome (BS) 2The abbreviations used are: BS, Bloom's syndrome; SCE, sister-chromatid exchange; ATPγS, adenosine 5′-O-(thiotriphosphate); ssDNA, single-stranded DNA; nt, nucleotide. helicase, BLM, has been implicated in the repair of damaged replication forks (14Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (578) Google Scholar). BLM is a member of the highly conserved RecQ family of DNA helicases. The family is named after the E. coli prototypical member RecQ, and members are defined by the presence of a conserved catalytic helicase domain (15Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (312) Google Scholar). Several RecQ helicases also contain additional identifiable motifs, known as the RQC and HRDC domains (15Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (312) Google Scholar). The precise function of these domains is unknown, although they have been implicated in the binding of proteins and/or DNA structures (16Bernstein D.A. Keck J.L. Structure (Camb.). 2005; 13: 1173-1182Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (216) Google Scholar, 18Brosh Jr., R.M. von Kobbe C. Sommers J.A. Karmakar P. Opresko P.L. Piotrowski J. Dianova I. Dianov G.L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar, 19Liu Z. Macias M.J. Bottomley M.J. Stier G. Linge J.P. Nilges M. Bork P. Sattler M. Structure (Lond.). 1999; 7: 1557-1566Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 20Morozov V. Mushegian A.R. Koonin E.V. Bork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 21von Kobbe C. Karmakar P. Dawut L. Opresko P. Zeng X.M. Brosh R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 22035-22044Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 22Wu L. Lung Chan K. Ralf C. Bernstein D.A. Garcia P.L. Bohr V.A. Vindigni A. Janscak P. Keck J.L. Hickson I.D. EMBO J. 2005; 24: 2679-2687Crossref PubMed Scopus (138) Google Scholar). In all organisms, RecQ helicases are essential for the maintenance of genome stability with many RecQ helicase mutants displaying replication defects and elevated levels of recombination events (14Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (578) Google Scholar). Indeed, one of the defining characteristics of BS cells is an increased frequency of sister chromatid exchanges (SCEs) (23Chaganti R.S. Schonberg S. German J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4508-4512Crossref PubMed Scopus (786) Google Scholar), which are thought to arise from the aberrant repair of damaged replication forks (14Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (578) Google Scholar). The BLM protein has been shown to contain three distinct biochemical activities as follows: a 3′-5′ DNA unwinding activity, a Holliday junction branch migration activity, and a single-stranded DNA annealing activity (24Cheok C.F. Wu L. Garcia P.L. Janscak P. Hickson I.D. Nucleic Acids Res. 2005; 33: 3932-3941Crossref PubMed Scopus (130) Google Scholar, 25Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 26Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (421) Google Scholar, 27Machwe A. Xiao L. Groden J. Matson S.W. Orren D.K. J. Biol. Chem. 2005; 280: 23397-23407Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In what role and in which cellular context these activities function remain to be determined. Given that the process of fork regression involves the annealing of the nascent leading and lagging strands to extrude a fourth arm, which can be extended and thus stabilized by branch migration, one possibility is that the three known catalytic activities of BLM might coordinate to promote fork regression. Here we show that BLM can convert model substrates that mimic stalled replication forks into “chicken foot” structures containing regressed arms that are in excess of several hundred base pairs. Moreover, we show that this ability to regress replication forks is not a general feature of RecQ helicases, suggesting a potentially specific role for BLM in the regression of forks during replication re-start pathways. Proteins—BLM was expressed and purified as described previously (25Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). RecG, RecQ, and RusA were kind gifts from Drs. Peter McGlynn, James L. Keck, and Robert G. Lloyd, respectively. Topoisomerase I was purchased from Promega. N.BbvC IA, N.BbvC IB, and restriction enzymes were purchased from New England Biolabs. Fork Regression Assays—The replication fork substrate (10 pm) was incubated with the indicated concentration of BLM, RecG, or RecQ in buffer H (50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 2 mm MgCl2, 2 mm ATP, 1 mm dithiothreitol, and 100 μg/ml bovine serum albumin). Where indicated 1 μl (2-10 units) of wheat germ topoisomerase I was also included in the reactions. Unless otherwise indicated, reactions were incubated for 60 min at 37 °C and then quenched with 10 mm ATPγS and 125 ng of M13 ssDNA prior to the addition of 8 mm MgCl2 and 1 μl (2-20 units depending on the enzyme) of the indicated restriction enzyme. Reactions were incubated for a 60 min to were to and the were then on a were and to for analysis a and RusA Assays—The junction as described previously P. J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; PubMed Scopus Google Scholar), or were incubated with the indicated of RusA in buffer containing mm Tris-HCl, pH 1 mm dithiothreitol, bovine serum 10 mm MgCl2, at 37 °C for Reactions were by the addition of and 50 mm and with 1 for a 10 min at 37 were on a were and to for analysis a and Assays—The arm helicase as described previously P. J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; PubMed Scopus Google Scholar), was incubated with either BLM or RecQ, at the indicated in buffer H at 37 °C for were on were and to for analysis a and of a events that occur during replication fork we developed a for the of of a stalled replication as in This involves the annealing of a to a via a single-stranded gap in and a single-stranded gap in The annealing a which can be to a by of the DNA with topoisomerase I. This in the of the The resulting a mimic of a replication fork in which the of the is a number of that for the process of replication fork regression. the of the arms the are fork regression to occur by the of DNA at the the substrate that not exist in the ability to the of the single-stranded gaps on of the of analysis of the that different of nascent strands at the fork might have on fork regression of the of are in and Regression of the fork in result in the of restriction from arms the into the regressed arm of the fork regression can be by the restriction of the of as a fragment of a that is on the restriction used In the the in associated with the of the gaps in and the fork in substrate a stalled replication fork in that is a single-stranded gap on the leading strand template, a that arise the of leading and lagging strand synthesis and The gap also to fork regression. we a in which the single-stranded gap on the leading strand template is as to the in the and of the of the of the strands at the fork of these strands be to anneal and result in fork regression. and were with to the presence of a regressed of with of the as a fragment that of the forks in substrate fork regression In a fragment was not of This that the gap on fork regression and that the of the in is in a fork of with either or are to the of also not of as to arise from of regressed arms and that the of the by of was of of the fork into a Holliday we with the Holliday RusA Lloyd R.G. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). we with which at a site in both of to a replication fork that arms of This to the branch in of the regressed fork by RusA in the substrate be to a fragment of a we also incubated RusA with a Holliday junction that was from and of The junction was of a homologous which a the site for RusA Lloyd R.G. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). RusA was to to of was by RusA to This indicated that of the forks in were in a Holliday junction a with the of as the fragment of with and In to of was by that the gap on the leading strand in fork regression establish that the fork in could be to a Holliday we with RecG, which has a known ability to promote fork regression (9McGlynn P. Lloyd R.G. Cell. 2000; 101: 35-45Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). RecG was to the of into regressed which could be with or to the that were to arise from of the regressed arm and these data that of the forks in are in a fork and that these forks can be regressed by the fork regression activity of BLM a Fork to a BLM could regress the fork in into a Holliday BLM was incubated with substrate were then with or to the presence of regressed BLM was in a to convert forks in the substrate into Holliday as by the restriction of were to by RecG and data not The of the was on the presence of ATP, a for the helicase and/or branch migration activity of BLM and the concentration of BLM of a regressed arm Given that of the substrate Holliday we that BLM can regressed arms from a replication fork structure. the of fork regression required the of the substrate with restriction was that the as a of BLM on the substrate by a process unrelated to fork regression. this reactions were quenched by addition of 10 mm ATPγS and 125 ng of M13 single-stranded DNA to of BLM prior to the addition of could be ATPγS and M13 ssDNA were with BLM but not at the of the with BLM 2 and This result that ATPγS and M13 ssDNA as of BLM and that the as a result of of and not the of BLM on establish that the as a result of fork the was to be and to contain the restriction for and which are the site and the of the gap in of the with either or rise to of or bp in with the and as a result of fork regression BLM to >250 bp in we the to which BLM could promote fork regression on regressed arms of bp could be BLM and and However, forks regressed to a site into the regressed arm, which is bp to the site and be to a fragment with and and Fork regression is to be a process in which the of the regressed arm be to protein concentration and/or however, although the of that regressed forks was to the concentration of BLM, the of regressed arm was BLM that is, for concentration of BLM, all regressed forks a regressed arm of bp and This suggests that BLM in a and not a manner to regress stalled replication that BLM-mediated fork regression is a the of fork regression was either the or which are bp in the regressed arm a concentration of BLM, the restriction site for was into the regressed arm min prior to the site for This as fork regression by BLM is a that at the fork. BLM-mediated Fork Regression of the of DNA can fork regression in L. C. C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). was that BLM not at the replication fork in substrate to promote regression. of the of BLM, through ability to could the substrate that cause fork regression. is required for BLM to promote fork we BLM on that been by to the of DNA that might arise of of BLM the substrate BLM was to regress the fork in This that that might arise is not the for BLM-mediated fork regression and that the of is not the mechanism by which BLM fork regression. that is not required for BLM-mediated fork regression was by the that the of wheat germ topoisomerase which both and not the ability of BLM to regress forks Fork Regression a of RecQ can or process a of and DNA which are also substrates for helicases (15Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (312) Google Scholar, 26Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (421) Google Scholar, P. J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; PubMed Scopus Google Scholar, H. J.K. Hickson I.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these is E. coli RecQ, which the three domains helicase, and HRDC that are in many RecQ family helicases, BLM (15Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (312) Google Scholar). RecQ has also been implicated in the unwinding of the lagging strand of stalled replication forks in vivo J. Mol. Genet. 1999; PubMed Scopus Google Scholar, T. T. H. H. Genes Dev. 2004; 18: PubMed Scopus Google Scholar). However, E. coli RecQ was not to fork regression on at at which BLM fork regression and and Moreover, at of RecQ BLM RecQ was to fork regression not of helicase activity a substrate indicated at RecQ an helicase activity BLM and the possibility that RecQ helicase activity might be to fork we used of RecQ and BLM that helicase activity, as by the arm these RecQ was to fork regression with The of replication forks is a in all and several have been proposed to how replication might be from a stalled fork Here we have shown that BLM is of the regression of model stalled replication forks in This novel activity of BLM at the replication fork and the fork in a manner for of >250 Regression to this is with the of structures in replication purified from cells to (12Sogo J.M. Lopes M. Foiani M. Science. 2002; 297: 599-602Crossref PubMed Scopus (678) Google Scholar, M. Foiani M. J.M. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism by which BLM fork regression of to which of the helicase, branch migration, and strand annealing activities of BLM is required and how they coordinate to fork regression. in which the nascent strands are by the helicase activity of BLM, by subsequent annealing to is fork regression could not be with E. coli RecQ, which can lagging strands at replication forks T. T. H. H. Genes Dev. 2004; 18: PubMed Scopus Google Scholar). The of RecQ to fork regression suggests that this activity might be a specific activity of BLM and not an function of RecQ helicases. The of the BLM and RecQ is that the of BLM are required for fork regression. However, the HRDC which is conserved in BLM, RecQ, and several RecQ family helicases, has been shown to the binding and unwinding activities of the protein in which and has been implicated in members of the RecQ helicase family (16Bernstein D.A. Keck J.L. Structure (Camb.). 2005; 13: 1173-1182Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Wu L. Lung Chan K. Ralf C. Bernstein D.A. Garcia P.L. Bohr V.A. Vindigni A. Janscak P. Keck J.L. Hickson I.D. EMBO J. 2005; 24: 2679-2687Crossref PubMed Scopus (138) Google Scholar). to which conserved and of BLM are important for fork regression. The cellular feature of BS cells is an elevated frequency of which are by homologous recombination E. C. M. S. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). One proposed mechanism by which BLM is through the of Holliday into L. Hickson I.D. 2003; PubMed Scopus Google Scholar). BLM-mediated is on the type topoisomerase which a with BLM for cellular L. Hickson I.D. 2003; PubMed Scopus Google Scholar, L. H. H. Hickson I.D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). this a BLM protein that the conserved domain is to suppress the elevated in BS cells P. L. Mol. Genet. 2001; PubMed Google Scholar). under BLM and in to V.A. L. C.F. Hickson I.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). These suggest that BLM may function in both and to One possibility is that fork regression by BLM the substrates utilized in both of these pathways. template and the regressed fork may be to the fork by the branch migration activity of BLM and as previously J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (421) Google Scholar). the regressed arm may be utilized by the recombination repair to replication downstream of the fork generating a Holliday junction and This is a model BLM will be at the site of the stalled fork and will be to the Holliday junction junction can the Holliday into rise to and The presence of BLM may also from the Holliday junction from fork regression and thus generating a strand BLM-mediated fork regression may be important in stalled forks In the of BLM, stalled forks may be to strand by such as the and recombination in BS cells (23Chaganti R.S. Schonberg S. German J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4508-4512Crossref PubMed Scopus (786) Google Scholar, G. I. M. H. H. A. Nat. Genet. 2000; PubMed Scopus Google Scholar). In RecQ helicases exist in addition to Where the of RecQ helicase mutants indicate both and for RecQ helicases E. M. H. G. Mol. Cell. Biol. 2005; PubMed Scopus (108) Google Scholar, M. T. A. M. S. H. T. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). In with BS elevated are also a feature of in and cells E. M. H. G. Mol. Cell. Biol. 2005; PubMed Scopus (108) Google Scholar, M. T. A. M. S. H. T. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). although fork regression might be an activity specific for BLM, to be RecQ helicases could also promote fork regression that might in either or to BLM to members of the and for Drs. Peter McGlynn, James L. Keck, and Robert G. Lloyd for RecG, and RusA respectively. also Peter for of the with