Title: Junction of RecQ Helicase Biochemistry and Human Disease
Abstract: RecQ helicases are a family of conserved enzymes required for maintaining the genomic integrity, that function as suppressors of inappropriate recombination. Mutations in Escherichia coli RecQ and in the Saccharomyces cerevisiae RecQ homolog, Sgs1, result in an increased frequency of illegitimate recombination (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar). In humans, defects in three RecQ family proteins are associated with rare autosomal-recessive disorders characterized by genomic instability and increased cancer susceptibility. Mutations in WRN, BLM, and RECQ4 give rise to the disorders Werner syndrome (WS), 1The abbreviations used are: WS, Werner syndrome; BS, Bloom syndrome; RTS, Rothmund-Thomson syndrome; ss, single strand; HJ, Holliday junction; HR, homologous recombination; NHEJ, non-homologous end joining; DSB, double strand break; SSB, single strand break; RPA, replication protein A; BER, base excision repair; ALT, alternative lengthening of telomeres; RQC, RecQ C-terminal region; HRDC, helicase RNase D C-terminal domain; PK, protein kinase. Bloom syndrome (BS), and Rothmund-Thomson syndrome (RTS), respectively, the clinical features of which have been reviewed elsewhere (2Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Google Scholar). Briefly, BS patients are predisposed to many types of cancer with the mean age of onset of 24. WS patients are especially predisposed to sarcomas, premature aging, and age-associated diseases. RTS patients have a characteristic rash, poikiloderma, and are predisposed to osteosarcomas and some features of premature aging. The molecular basis of genomic instability and premature aging is not well understood. The RecQ family is named after E. coli RecQ helicase, a well characterized prototypical member (Fig. 1). Helicases separate complementary strands of nucleic acids in a reaction coupled to NTP hydrolysis. RecQ helicases have a common helicase domain, which binds and hydrolyzes ATP. Most RecQ helicases have a highly conserved multifunctional RecQ C-terminal region (RQC) and a helicase RNase D C-terminal (HRDC) domain (Fig. 1). A recent report of the x-ray crystal structure for the E. coli RecQ catalytic core indicates that the RQC domain contains DNA and protein binding motifs (3Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Google Scholar). Consistent with this, the RQC domain of WRN binds to various DNA substrates and mediates interactions with other proteins involved in DNA metabolism (4von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Google Scholar). The E. coli RecQ HRDC domain is required for stable DNA binding but not for catalytic activity (5Bernstein D.A. Keck J.L. Nucleic Acids Res. 2003; 31: 2778-2785Google Scholar). Similarly, the HRDC domain of human WRN also binds DNA substrates but is not required for catalytic activity (4von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Google Scholar). Two RecQ family proteins WRN and Xenopus laevis FFA-1 also have an exonuclease domain. Bacteria and yeast have a single RecQ family member, and up to five RecQ members have been found in mammals. To better define the precise roles of RecQ helicases in vivo, significant research effort has been devoted to characterizing the biochemical properties of RecQ helicases and to identifying important protein interactions between RecQ helicases and other well characterized proteins. This review will focus primarily on these aspects and on the most well characterized RecQ helicases, due to space limitations. RecQ helicase genetic studies in yeast have been recently reviewed elsewhere (6Khakhar R.R. Cobb J.A. Bjergbaek L. Hickson I.D. Gasser S.M. Trends Cell Biol. 2003; 13: 493-501Google Scholar). All RecQ helicases purified and characterized to date unwind duplex DNA in a 3′ to 5′ direction with respect to the DNA strand bound by the helicase. Substrate preferences are determined by comparing product amounts, reaction kinetics, and/or protein affinities for the substrates in side-by-side reactions. E. coli RecQ has broad DNA substrate specificity and acts on DNA duplexes containing blunt or forked termini, duplexes with 3′ or 5′ single strand (ss) tails, D-loops, and 3- or 4-way (Holliday) junctions (7Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Google Scholar) (Fig. 2). Mammalian and yeast RecQ helicases are less promiscuous than E. coli RecQ, which may reflect the DNA binding specificities of their unique N- and C-terminal protein domains. S. cerevisiae Sgs1 and human BLM and WRN do not unwind duplexes with blunt termini or with 5′ ssDNA tails (Fig. 2), but these enzymes preferentially unwind forked duplexes with branched structures or junctions (8Bennett R.J. Keck J.L. Wang J.C. J. Mol. Biol. 1999; 289: 235-248Google Scholar, 9Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Scopus (486) Google Scholar). WRN and BLM recognize and specifically bind to junction sites and have higher relative affinity for substrates with junctions (10Shen J.C. Loeb L.A. Nucleic Acids Res. 2000; 28: 3260-3268Google Scholar, 11Orren D.K. Theodore S. Machwe A. Biochemistry. 2002; 41: 13483-13488Google Scholar, 12van Brabant A.J. Ye T. Sanz M. German III, J.L. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Google Scholar, 13Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Google Scholar). They also preferentially unwind a Holliday junction (HJ) substrate with short arms constructed from oligonucleotides, compared with a forked duplex (9Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Scopus (486) Google Scholar), and promote extensive branch migration (several kilobases) of long-arm HJ substrates generated by RecA protein (α-structure) (2Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Google Scholar). The latter is particularly remarkable because WRN and BLM are normally low to moderately processive (<100 bp). Both enzymes unwind a blunt duplex interrupted by an internal bubble, but small bubbles (4 nucleotides) are not unwound (9Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Scopus (486) Google Scholar). WRN preferentially binds a D-loop (Fig. 2), compared to a simple bubble, and releases the invading strand of a D-loop whether it has a 3′- or 5′-protruding tail or no tail (11Orren D.K. Theodore S. Machwe A. Biochemistry. 2002; 41: 13483-13488Google Scholar). BLM favors a D-loop with a 3′-protruding tail, but all D-loop forms tested are unwound more efficiently than a bubble, similar to WRN (12van Brabant A.J. Ye T. Sanz M. German III, J.L. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Google Scholar). RecQ helicases appear to favor substrates that mimic recombination and replication intermediates. To determine whether all eukaryotic RecQ helicases share common substrate specificities will require characterization of the other family members. Similar to WRN, BLM, and Sgs1, the small isoform of DmRecQ5 helicase from Drosophila melanogaster (14Ozsoy A.Z. Ragonese H.M. Matson S.W. Nucleic Acids Res. 2003; 31: 1554-1564Google Scholar) and human RecQ1 (RecQL) (15Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Google Scholar) are inactive on 5′ ssDNA tailed or blunt-ended duplexes but do unwind some junction-containing substrates (Fig. 2). In contrast, DmRecQ5 preferentially unwinds forked duplexes and is less active on HJ and bubble substrates (14Ozsoy A.Z. Ragonese H.M. Matson S.W. Nucleic Acids Res. 2003; 31: 1554-1564Google Scholar). RecQ helicases likely have some complementary and distinct roles in vivo. RecQ helicases also unwind non-canonical (non-B form) DNA helical structures. The genome contains a variety of sequences that can form DNA triple helices and G-quadruplex structures that may block DNA metabolic pathways. Triplexes occur when a third strand forms stable sequence-specific interactions with the major groove of duplex DNA and quadrahelices form in G-rich sequences; both are stabilized by Hoogsteen base pairing. G-quadruplexes form readily under physiological conditions in vitro in telomeric DNA, Fragile X syndrome repeat sequences, and immunoglobulin switch regions (16Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Google Scholar, 17Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Google Scholar). BLM, WRN, and Sgs1 unwind G-quadruplex structures with a 3′ ssDNA tail, and several studies show a remarkable preference for G-quadruplex substrates relative to junction-containing substrates (16Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Google Scholar, 17Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Google Scholar). Huber et al. (18Huber M.D. Lee D.C. Maizels N. Nucleic Acids Res. 2002; 30: 3954-3961Google Scholar) found that BLM and Sgs1 preferentially bound to, and unwound, G-quadruplexes relative to a HJ constructed from oligonucleotides. Similarly, BLM and WRN unwind triplex DNA with a 3′ ssDNA tail more efficiently than a duplex with a 3′ ssDNA tail (19Brosh Jr., R.M. Majumdar A. Desai S. Hickson I.D. Bohr V.A. Seidman M.M. J. Biol. Chem. 2001; 276: 3024-3030Google Scholar). G-quadruplex and triplex DNA have been implicated in DNA rearrangements including deletions, sister chromatid exchange, and homologous and illegitimate recombination (19Brosh Jr., R.M. Majumdar A. Desai S. Hickson I.D. Bohr V.A. Seidman M.M. J. Biol. Chem. 2001; 276: 3024-3030Google Scholar). Roles for RecQ helicases in disrupting such structures are consistent with the observed elevated genomic instability in RecQ-deficient cells. The above results suggest that human RecQ helicases prefer substrates that are DNA metabolic intermediates (Fig. 2), including forked and flap structures (replication and repair), bubbles (repair and transcription), D-loops and HJs (recombination), and G-quadruplex DNA and D-loops (associated with telomeric DNA). Thus, the in vitro biochemistry suggests that RecQ helicases may play accessory roles in DNA repair, recombination, replication, telomere processing, and transcription. There is accumulating cellular evidence for one or more of the RecQ helicases functioning in many aspects of these processes (see below). The WRN exonuclease is homologous to E. coli RNase D. Thus far, WRN is the only mammalian RecQ helicase identified that contains an additional 3′- to 5′-exonuclease activity. Characterization of the 3′- to 5′-exonuclease in the X. laevis FFA-1 RecQ helicase remains to be determined. WRN degrades DNA exonucleolytically from the recessed 3′ end of a DNA duplex or DNA/RNA heteroduplex and is essentially inactive on ssDNA and duplex DNA with blunt ends or a recessed 5′ end (20Huang S. Beresten S. Li B. Oshima J. Ellis N.A. Campisi J. Nucleic Acids Res. 2000; 28: 2396-2405Google Scholar). Interestingly, WRN can degrade DNA starting from a blunt-ended DNA duplex, if the substrate contains a junction or alternate structure such as a fork, HJ, or D-loop (10Shen J.C. Loeb L.A. Nucleic Acids Res. 2000; 28: 3260-3268Google Scholar, 11Orren D.K. Theodore S. Machwe A. Biochemistry. 2002; 41: 13483-13488Google Scholar), possibly reflecting the high affinity of WRN for such structures. Although the WRN exonuclease and helicase prefer similar substrates, it is not clear whether both activities contribute to the processing of a DNA substrate in a common molecular pathway. In vitro, WRN helicase and exonuclease act simultaneously at opposite ends of a long DNA forked duplex and cooperate to separate the strands (21Opresko P.L. Laine J.P. Brosh Jr., R.M. Seidman M.M. Bohr V.A. J. Biol. Chem. 2001; 276: 44677-44687Google Scholar). We recently observed a similar cooperation in removing the invading strand of a long D-loop. 2P. L. Opresko and V. A. Bohr, unpublished data. Recent evidence indicates that an appropriate balance between the WRN helicases and exonuclease activity is important for optimal repair via homologous recombination (22Chen L. Huang S. Lee L. Davalos A. Schiestl R.H. Campisi J. Oshima J. Aging Cell. 2003; 2: 191-199Google Scholar). The WRN exonuclease also acts on some mismatched base pairs (23Kamath-Loeb A.S. Shen J.C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Google Scholar) and certain modified base pairs including uracil and hypoxanthine, whereas other lesions, such as apurinic sites or 8-oxoguanine, inhibit the WRN exonuclease (24Machwe A. Ganunis R. Bohr V.A. Orren D.K. Nucleic Acids Res. 2000; 28: 2762-2770Google Scholar). Therefore, the WRN exonuclease may repair DNA termini produced as intermediates during DNA replication, repair, and/or recombination. WRN may also participate in a DNA surveillance system that senses DNA damage and targets it for repair. Evidence indicates that RecQ helicases may exist in several different oligomeric states. Hexameric rings of full-length BLM (25Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Google Scholar) and trimers of WRN (20Huang S. Beresten S. Li B. Oshima J. Ellis N.A. Campisi J. Nucleic Acids Res. 2000; 28: 2396-2405Google Scholar) were observed with size exclusion chromatography and/or by electron microscopy. However, the oligomeric state of these enzymes may be influenced by substrate binding, catalytic state, protein interactions, and/or post-translational modifications. E. coli RecQ forms monomers in solution and unwinds DNA and hydrolyzes ATP as a monomer (26Xu H.Q. Deprez E. Zhang A.H. Tauc P. Ladjimi M.M. Brochon J.C. Auclair C. Xi X.G. J. Biol. Chem. 2003; 278: 34925-34933Google Scholar). Janscak et al. (27Janscak P. Garcia P.L. Hamburger F. Makuta Y. Shiraishi K. Imai Y. Ikeda H. Bickle T.A. J. Mol. Biol. 2003; 330: 29-42Google Scholar) recently purified a BLM fragment (amino acids 642–1290), containing the conserved helicase, RQC, and HRDC domains, that forms monomers in solution when it is catalytically active and/or bound to DNA. This monomeric fragment retains the substrate specificity of full-length BLM, indicating that the hexameric form of full-length BLM is not required for activity. We have recently purified a WRN fragment that consists primarily of the helicase domain (amino acids 400–946) and retains helicase activity (28Harrigan J.A. Opresko P.L. von Kobbe C. Kedar P.S. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 2003; 278: 22686-22695Google Scholar), although the oligomeric state remains to be determined. So far, evidence suggests that human RecQ helicases may dynamically transition between several possible oligomeric states. This conformational flexibility may provide an additional mechanism for regulating activity and function of RecQ helicases. Consistent with their ability to act on multiple intermediates in DNA processing, RecQ helicases interact with many other proteins involved in DNA metabolism. Therefore, it has been difficult to accurately infer their specific biological functions through the identification of protein-binding partners. A biochemical approach to addressing this problem has been to determine which protein partners also modulate the biochemical activity of the interacting enzymes. This review focuses specifically on this class of protein partners in the context of the molecular pathways. RecQ helicases are proposed to function during DNA replication in restoring stalled or broken replication forks, such as when the fork encounters blocking lesions or strand breaks. Homologous recombination (HR) is involved in replication fork restart and repair (6Khakhar R.R. Cobb J.A. Bjergbaek L. Hickson I.D. Gasser S.M. Trends Cell Biol. 2003; 13: 493-501Google Scholar). RecQ helicases resolve a variety of recombination intermediates (Fig. 2) and are proposed to prevent inappropriate DNA strand crossovers during replication restart (for a review of models see Refs. 1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar and 2Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Google Scholar). In E. coli, RecQ and RecJ degrade the nascent lagging strand at UV-induced stalled DNA replication forks to promote fork stabilization and suppress inappropriate recombination (29Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar). In yeast, Sgs1 is required for stabilization of stalled replication forks induced by hydroxyurea treatment (30Cobb J.A. Bjergbaek L. Shimada K. Frei C. Gasser S.M. EMBO J. 2003; 22: 4325-4336Google Scholar). Roles for WRN and BLM in replication fork stabilization are less clear; however, they both interact with components of the replication fork. WRN and BLM interact physically and functionally with FEN-1 endonuclease (31Brosh 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: 5791-5801Google Scholar, 32Sharma S. Sommers J.A. Wu L. Bohr V.A. Hickson I.D. Brosh Jr., R.M. J. Biol. Chem. 2004; 279: 9847-9856Google Scholar), an enzyme that acts in the processing of Okazaki fragments during lagging strand DNA replication. In addition, WRN and BLM co-localize with replication protein (RPA) after DNA damage (33Constantinou A. Tarsounas M. Karow J.K. Brosh R.M. Bohr V.A. Hickson I.D. West S.C. EMBO Rep. 2000; 1: 80-84Google Scholar, 34Sakamoto S. Nishikawa K. Heo S.J. Goto M. Furuichi Y. Shimamoto A. Genes Cells. 2001; 6: 421-430Google Scholar, 35Bischof O. Kim S.H. Irving J. Beresten S. Ellis N.A. Campisi J. J. Cell Biol. 2001; 153: 367-380Google Scholar), and RPA binds to WRN and BLM and stimulates their unwinding of long DNA duplexes (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar). RPA similarly stimulates human RecQ1 helicase (15Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Google Scholar). Furthermore, cells from BS and WS patients are characterized by defects in DNA replication consistent with the inability to properly recover from DNA replication fork demise (2Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Google Scholar). Thus, RecQ helicases may function with protein partners in the processing of DNA intermediates during replication fork recovery. Alternatively, RecQ helicases may remove blocking DNA structures directly, such as G-quadruplexes, which are preferred substrates for these enzymes (Fig. 2). WRN interacts with replicative DNA polymerase δ and promotes bypass of hairpin and G-quadruplex structures (36Kamath-Loeb A.S. Loeb L.A. Johansson E. Burgers P.M. Fry M. J. Biol. Chem. 2001; 276: 16439-16446Google Scholar). RecQ helicases may also resolve blocks due to excessive torsional stress induced by DNA replication or repair, via a conserved interaction with topoisomerases (from E. coli to humans) (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar). BLM binds to and stimulates the α-isoform of human topoisomerase III (37Wu L. Hickson I.D. Nucleic Acids Res. 2002; 30: 4823-4829Google Scholar), and WRN binds to and stimulates human topoisomerase I (38Laine J.P. Opresko P.L. Indig F.E. Harrigan J.A. von Kobbe C. Bohr V. Cancer Res. 2003; 63: 7136-7146Google Scholar). These blocks could contribute to replication fork stalling. RecQ helicases are also proposed to function in HR to promote proper intermediate resolution and suppress strand crossover events. HR is important for repairing chromosomal double strand breaks (DSB) that can result during replication when the fork encounters a single strand break (SSB) or gap. BS cells and yeast sgs1 mutants display a high frequency of HR-mediated sister chromatid exchanges, which likely result from DSBs during DNA replication (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar). Recent experiments indicate that HR-mediated strand crossover events are suppressed by Sgs1 and Top3 in yeast (39Ira G. Malkova A. Liberi G. Foiani M. Haber J.E. Cell. 2003; 115: 401-411Google Scholar) and by the action of BLM and TopIIIα during the resolution of double HJs in vitro (40Wu L. Hickson I.D. Nature. 2003; 426: 870-874Google Scholar). WS and RTS cells display an increased frequency of chromosomal rearrangements, including translocations and deletions (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar, 41Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Google Scholar), which may result partly from DSBs. RecQ helicases interact with Rad51 and Rad52 proteins, among other proteins involved in HR, and likely contribute to proper resolution of intermediates and/or prevention of illegitimate recombination. Rad51 nucleates onto ssDNA via an interaction with Rad52 and facilitates strand invasion during recombination (42Sung P. Krejci L. Van Komen S. Sehorn M.G. J. Biol. Chem. 2003; 278: 42729-42732Google Scholar). WRN and BLM co-localize to DNA damage-induced Rad51 foci (34Sakamoto S. Nishikawa K. Heo S.J. Goto M. Furuichi Y. Shimamoto A. Genes Cells. 2001; 6: 421-430Google Scholar, 35Bischof O. Kim S.H. Irving J. Beresten S. Ellis N.A. Campisi J. J. Cell Biol. 2001; 153: 367-380Google Scholar, 43Wu L. Davies S.L. Levitt N.C. Hickson I.D. J. Biol. Chem. 2001; 276: 19375-19381Google Scholar), and both BLM and yeast Sgs1 physically bind Rad51 (43Wu L. Davies S.L. Levitt N.C. Hickson I.D. J. Biol. Chem. 2001; 276: 19375-19381Google Scholar). Suppression of recombination in some RecQ-deficient cells improves cell survival, suggesting that toxic recombination intermediates arise and persist in the absence of RecQ helicases. In yeast, deletion of Sgs1 and Srs2 helicases results in synthetic lethality that is rescued by mutations in RAD51 or RAD52 (44Gangloff S. Soustelle C. Fabre F. Nat. Genet. 2000; 25: 192-194Google Scholar). Similarly, the expression of a dominant-negative Rad51 mutant suppresses recombination in WS cells and increases cell survival after DNA damage (45Saintigny Y. Makienko K. Swanson C. Emond M.J. Monnat Jr., J.R. Mol. Cell. Biol. 2002; 22: 6971-6978Google Scholar). In addition, WRN co-localizes and interacts with Rad52 and in vitro shows modest stimulation of Rad52-mediated ssDNA annealing (46Baynton K. Otterlei M. Bjoras M. von Kobbe C. Bohr V.A. Seeberg E. J. Biol. Chem. 2003; 278: 36476-36486Google Scholar). Biochemical and genetic data indicate that RecQ helicases likely function upstream and/or downstream of HR to properly dissociate recombination intermediates and to prevent inappropriate strand exchanges. However, potential roles in facilitating recombination cannot be ruled out. Roles for RecQ helicases may not be limited to S-phase and HR related pathways. Although BLM, RTS, and Sgs1 expression levels are highest in S-phase, WRN is constitutively expressed throughout the cell cycle (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar). Double strand breaks are repaired primarily via two pathways: HR, which predominates in S-phase, and the more error-prone non-homologous end-joining (NHEJ) pathway, which predominates during G1. WS cells show a mild to strong hypersensitivity to agents that cause DSBs: ionizing radiation and DNA cross-linking reagents, respectively (41Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Google Scholar). Interestingly, RTS cells are also hypersensitive to ionizing radiation (47Kerr B. Ashcroft G.S. Scott D. Horan M.A. Ferguson M.W. Donnai D. J. Med. Genet. 1996; 33: 928-934Google Scholar), but whether this relates to defects in HR or NHEJ is unknown. Essential components of the NHEJ pathway have been found to interact with WRN, namely Ku and DNA-PK. A search for protein interactions with the WRN C-terminal region identified the Ku heterodimer as the most prominent binder (48Cooper M.P. Machwe A. Orren D.K. Brosh R.M. Ramsden D. Bohr V.A. Genes Dev. 2000; 14: 907-912Google Scholar). Ku stimulates the WRN 3′- to 5′-exonuclease and increases the processivity of the enzyme (48Cooper M.P. Machwe A. Orren D.K. Brosh R.M. Ramsden D. Bohr V.A. Genes Dev. 2000; 14: 907-912Google Scholar, 49Karmakar P. Piotrowski J. Brosh Jr., R.M. Sommers J.A. Miller S.P. Cheng W.H. Snowden C.M. Ramsden D.A. Bohr V.A. J. Biol. Chem. 2002; 277: 18291-18302Google Scholar, 50Li B. Comai L. J. Biol. Chem. 2001; 276: 9896-9902Google Scholar). Ku is part of the DNA-PK kinase complex, and DNA-PK-dependent phosphorylation of WRN has been observed in vitro and in vivo and regulates the WRN helicase and exonuclease (49Karmakar P. Piotrowski J. Brosh Jr., R.M. Sommers J.A. Miller S.P. Cheng W.H. Snowden C.M. Ramsden D.A. Bohr V.A. J. Biol. Chem. 2002; 277: 18291-18302Google Scholar, 51Yannone S.M. Roy S. Chan D.W. Murphy M.B. Huang S. Campisi J. Chen D.J. J. Biol. Chem. 2001; 276: 38242-38248Google Scholar). Furthermore, aberrant products of NHEJ reactions have been observed in WS cells (22Chen L. Huang S. Lee L. Davalos A. Schiestl R.H. Campisi J. Oshima J. Aging Cell. 2003; 2: 191-199Google Scholar). Precise roles for WRN and potentially other RecQ helicases in NHEJ remain to be determined. Roles for RecQ helicases in repair of SSB have also been proposed. Several enzymes that interact with WRN, including RPA, FEN1, and DNA polymerase δ, function in long patch base excision repair (BER), an important process for repairing modified bases as well as SSBs. WRN binds to the primary BER enzyme, DNA polymerase β, and stimulates strand displacement DNA synthesis (28Harrigan J.A. Opresko P.L. von Kobbe C. Kedar P.S. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 2003; 278: 22686-22695Google Scholar). Another important BER enzyme, PARP-1, was recently reported to be the most prominent binder to the WRN RQC domain (52von Kobbe C. Harrigan J.A. May A. Opresko P.L. Cheng W.H. Bohr V.A. Mol. Cell. Biol. 2003; 23: 8601-8613Google Scholar). PARP-1 binds strongly to SSBs and acts in the DNA damage surveillance network, partly by ribosylating a variety of nuclear proteins in response to DNA damage. WS cells are deficient in poly(ADP-ribosyl)ation in response to H2O2 and methyl methanesulfonate (52von Kobbe C. Harrigan J.A. May A. Opresko P.L. Cheng W.H. Bohr V.A. Mol. Cell. Biol. 2003; 23: 8601-8613Google Scholar), indicating the SSBs may not be properly processed in WS cells. For example, in yeast the 3′ to 5′ Srs2 helicase shuttles SSBs formed during replication into gap-filling repair pathways as opposed to recombinogenic pathways (53Fabre F. Chan A. Heyer W.D. Gangloff S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16887-16892Google Scholar). Similarly, incomplete BER intermediates (small gaps) are shuttled to the HR pathway for resolution (54Sobol R.W. Kartalou M. Almeida K.H. Joyce D.F. Engelward B.P. Horton J.K. Prasad R. Samson L.D. Wilson S.H. J. Biol. Chem. 2003; 278: 39951-39959Google Scholar). Therefore, some RecQ helicases may function in proper repair of SSBs, perhaps by dissociating inappropriate recombination intermediates. Whether other RecQ helicases function in pathways other than HR outside of S-phase remains to be determined. Cellular and biochemical evidence also indicate a role for RecQ helicases in maintaining telomeric ends. For example, evidence supports the formation of D-loop and G-quadruplex DNA at telomeric ends (41Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Google Scholar), and these structures are strongly preferred substrates for WRN, BLM, and Sgs1 (see above). WRN and BLM interact physically and functionally with the critical telomere binding and maintenance protein TRF2, whereby TRF2 promotes their helicase activity on short duplexes (55Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Google Scholar). WRN and BLM co-localize with TRF2 in nuclear foci of immortalized human cell lines that use a telomerase-independent pathway to prevent telomere erosion, termed ALT (alternative lengthening of telomeres) (55Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Google Scholar, 56Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar, 57Stavropoulos D.J. Bradshaw P.S. Li X. Pasic I. Truong K. Ikura M. Ungrin M. Meyn M.S. Hum. Mol. Genet. 2002; 11: 3135-3144Google Scholar). An ALT pathway in S. cerevisiae is dependent on recombination proteins Rad52 and Rad50 and requires Sgs1 (56Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar, 58Huang P. Pryde F.E. Lester D. Maddison R.L. Borts R.H. Hickson I.D. Louis E.J. Curr. Biol. 2001; 11: 125-129Google Scholar, 59Cohen H. Sinclair D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3174-3179Google Scholar). ALT is poorly understood, but a highly regulated form of ALT may act to repair and protect telomeric ends in normal somatic cells that lack telomerase activity. Defects in telomere structure can initiate a DNA damage response and may lead to telomeric end fusions and chromosome breakage if not properly repaired (60De Lange T. Oncogene. 2002; 21: 532-540Google Scholar). Consistent with this, WS and BS cells display some cellular features associated with defects in telomere maintenance. For example, telomere dysfunction can lead to premature senescence, which is a characteristic of WS fibroblasts (41Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Google Scholar). These results are consistent with a possible role for RecQ helicases in repair and processing of telomeric end structures. RecQ helicases have been proposed to function in sensing and responding to DNA damage, especially during S-phase. Evidence in yeast indicates that Sgs1 participates in the S-phase checkpoint response to DNA damage (6Khakhar R.R. Cobb J.A. Bjergbaek L. Hickson I.D. Gasser S.M. Trends Cell Biol. 2003; 13: 493-501Google Scholar). In mammalian cells, WRN tyrosine phosphorylation is induced by bleomycin (γ-irradiation mimic) in a manner dependent on the c-Abl kinase DNA damage response pathway (61Cheng W.H. von Kobbe C. Opresko P.L. Fields K.M. Ren J. Kufe D. Bohr V.A. Mol. Cell. Biol. 2003; 23: 6385-6395Google Scholar). BLM is part of the BASC (BRCA1-associated genome surveillance complex) which contains important DNA damage response proteins including the ATM and ATR kinases and the S-phase checkpoint protein Nbs1 (62Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Google Scholar). BLM and WRN have been shown to be phosphorylated in an ATM- and ATR-dependent manner in response to replication fork stalling (2Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Google Scholar, 63Franchitto A. Pichierri P. Hum. Mol. Genet. 2002; 11: 2447-2453Google Scholar). Because BS cells are not hypersensitive to ionizing radiation, the role for BLM in suppressing crossover events is proposed to be important for HR associated with gaps that arise during DNA replication (40Wu L. Hickson I.D. Nature. 2003; 426: 870-874Google Scholar). In addition, the important DNA damage response protein p53 binds to BLM and WRN and inhibits their resolution of HJ substrates and the WRN exonuclease (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar). Furthermore, WRN and BLM co-localize with p53 in response to replication fork stalling (64Brosh J. Karmakar P. Sommers J.A. Yang Q. Wang X.W. Spillare E.A. Harris C.C. Bohr V.A. J. Biol. Chem. 2001; 276: 35093-35102Google Scholar, 65Sengupta S. Linke S.P. Pedeux R. Yang Q. Farnsworth J. Garfield S.H. Valerie K. Shay J.W. Ellis N.A. Wasylyk B. Harris C.C. EMBO J. 2003; 22: 1210-1222Google Scholar). Thus, RecQ helicases likely play an important role in the cellular response to DNA damage, particularly during S-phase. The results summarized in this review indicate that RecQ helicases are multifunctional and likely play a role in many facets of DNA metabolism. The complex biology of RecQ helicases presents a significant research challenge. One possible scenario is that RecQ helicases may function, in part, as transducers that act in DNA repair, replication, and recombination. In response to an upstream signal, transducers activate downstream partners, for example, by recruiting appropriate repair enzymes to specific sites of DNA damage. As mentioned, Sgs1 appears to function in the S-phase checkpoint pathway (6Khakhar R.R. Cobb J.A. Bjergbaek L. Hickson I.D. Gasser S.M. Trends Cell Biol. 2003; 13: 493-501Google Scholar, 30Cobb J.A. Bjergbaek L. Shimada K. Frei C. Gasser S.M. EMBO J. 2003; 22: 4325-4336Google Scholar). BLM may act early in response to DNA damage during S-phase, because BLM was recently reported to be required for efficient localization of protein factors to repair complexes/foci after replication fork stalling (65Sengupta S. Linke S.P. Pedeux R. Yang Q. Farnsworth J. Garfield S.H. Valerie K. Shay J.W. Ellis N.A. Wasylyk B. Harris C.C. EMBO J. 2003; 22: 1210-1222Google Scholar, 66Davalos A.R. Campisi J. J. Cell Biol. 2003; 162: 1197-1209Google Scholar, 67Franchitto A. Pichierri P. J. Cell Biol. 2002; 157: 19-30Google Scholar). In addition, recent evidence indicates that WRN may function as a structural scaffold protein in NHEJ (22Chen L. Huang S. Lee L. Davalos A. Schiestl R.H. Campisi J. Oshima J. Aging Cell. 2003; 2: 191-199Google Scholar). DNA damage-induced SSBs or DSBs, collapsed replication forks, or dysfunctional telomeres can initiate a DNA damage response, and many of these structures are likely to interact with RecQ helicase proteins in vivo. RecQ helicases may be able to shuttle DNA damage, perhaps through processing of intermediates, to the appropriate DNA repair pathways such as BER/SSB repair, HR, NHEJ, or gap-filling (Fig. 3). This may be more relevant to multicellular organisms, which evolved multiple RecQ helicase variants that may have specialized roles, perhaps for pathways outside of S-phase.