Junction of RecQ Helicase Biochemistry and Human Disease*

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 (1). In hu- mans, defects in three RecQ family proteins are associated with rare autosomal-recessive disorders characterized by genomic insta- bility and increased cancer susceptibility. Mutations in WRN, BLM, and RECQ4 give rise to the disorders Werner syndrome (WS), 1 Bloom syndrome (BS), and Rothmund-Thomson syndrome (RTS), respectively, the clinical features of which have been re- viewed elsewhere (2). 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 instabil- ity 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

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 (1). 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), 1 Bloom syndrome (BS), and Rothmund-Thomson syndrome (RTS), respectively, the clinical features of which have been reviewed elsewhere (2). 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 ageassociated 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 (3). Consistent with this, the RQC domain of WRN binds to various DNA substrates and mediates interactions with other proteins involved in DNA metabolism (4). The E. coli RecQ HRDC domain is required for stable DNA binding but not for catalytic activity (5). Similarly, the HRDC domain of human WRN also binds DNA substrates but is not required for catalytic activity (4). 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 (6).

Biochemical Characteristics of RecQ Helicases
All RecQ helicases purified and characterized to date unwind duplex DNA in a 3Ј to 5Ј 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 3Ј or 5Ј single strand (ss) tails, D-loops, and 3-or 4-way (Holliday) junctions (7) (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 5Ј ssDNA tails (Fig. 2), but these enzymes preferentially unwind forked duplexes with branched structures or junctions (8,9). WRN and BLM recognize and specifically bind to junction sites and have higher relative affinity for substrates with junctions (10 -13). They also preferentially unwind a Holliday junction (HJ) substrate with short arms constructed from oligonucleotides, compared with a forked duplex (9), and promote extensive branch migration (several kilobases) of long-arm HJ substrates generated by RecA protein (␣-structure) (2). The latter is particularly remarkable because WRN and BLM are normally low to moderately processive (Ͻ100 bp). Both enzymes unwind a blunt duplex interrupted by an internal bubble, but small bubbles (4 nucleotides) are not unwound (9). 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 3Ј-or 5Ј-protruding tail or no tail (11). BLM favors a D-loop with a 3Ј-protruding tail, but all D-loop forms tested are unwound more efficiently than a bubble, similar to WRN (12). 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 (14) and human RecQ1 (RecQL) (15) are inactive on 5Ј 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 (14). RecQ helicases likely have some complementary and distinct roles in vivo.

G-quadruplex and Triplex DNA Substrates
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 (16,17). BLM, WRN, and Sgs1 unwind G-quadruplex structures with a 3Ј ssDNA tail, and several studies show a remarkable preference for Gquadruplex substrates relative to junction-containing substrates (16,17). Huber et al. (18) 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 3Ј ssDNA tail more efficiently than a duplex with a 3Ј ssDNA tail (19). G-quadruplex and triplex DNA have been implicated in DNA rearrangements including deletions, sister chromatid exchange, and homologous and illegitimate recombination (19). 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 Gquadruplex 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).

WRN Exonuclease Activity
The WRN exonuclease is homologous to E. coli RNase D. Thus far, WRN is the only mammalian RecQ helicase identified that contains an additional 3Ј-to 5Ј-exonuclease activity. Characterization of the 3Ј-to 5Ј-exonuclease in the X. laevis FFA-1 RecQ helicase remains to be determined. WRN degrades DNA exonucleolytically from the recessed 3Ј end of a DNA duplex or DNA/RNA heteroduplex and is essentially inactive on ssDNA and duplex DNA with blunt ends or a recessed 5Ј end (20). 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 (10,11), 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 oppo-site ends of a long DNA forked duplex and cooperate to separate the strands (21). We recently observed a similar cooperation in removing the invading strand of a long D-loop. 2 Recent evidence indicates that an appropriate balance between the WRN helicases and exonuclease activity is important for optimal repair via homologous recombination (22).
The WRN exonuclease also acts on some mismatched base pairs (23) and certain modified base pairs including uracil and hypoxanthine, whereas other lesions, such as apurinic sites or 8-oxoguanine, inhibit the WRN exonuclease (24). 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.

RecQ Helicase Oligomeric State
Evidence indicates that RecQ helicases may exist in several different oligomeric states. Hexameric rings of full-length BLM (25) and trimers of WRN (20) 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 (26). Janscak et al. (27) 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 (28), 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.

Putative Roles for RecQ Helicases with Protein Partners
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.

Roles in DNA Replication
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 (6). 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. 1 and 2). 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 (29). In yeast, Sgs1 is required for stabilization of stalled replication forks induced by hydroxyurea treatment (30). 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 (31,32), 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 (33)(34)(35), and RPA binds to WRN and BLM and stimulates their unwinding of long DNA duplexes (1). RPA similarly stimulates human RecQ1 helicase (15). 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 (2). Thus, RecQ 2 P. L. Opresko and V. A. Bohr, unpublished data. 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 ␦ and promotes bypass of hairpin and G-quadruplex structures (36). 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) (1). BLM binds to and stimulates the ␣-isoform of human topoisomerase III (37), and WRN binds to and stimulates human topoisomerase I (38). These blocks could contribute to replication fork stalling.

Roles in Recombination
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 (1). Recent experiments indicate that HR-mediated strand crossover events are suppressed by Sgs1 and Top3 in yeast (39) and by the action of BLM and TopIII␣ during the resolution of double HJs in vitro (40). WS and RTS cells display an increased frequency of chromosomal rearrangements, including translocations and deletions (1,41), 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 (42). WRN and BLM co-localize to DNA damage-induced Rad51 foci (34,35,43), and both BLM and yeast Sgs1 physically bind Rad51 (43). 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 (44). Similarly, the expression of a dominant-negative Rad51 mutant suppresses recombination in WS cells and increases cell survival after DNA damage (45). In addition, WRN co-localizes and interacts with Rad52 and in vitro shows modest stimulation of Rad52-mediated ssDNA annealing (46). 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 in Other DNA Repair Pathways
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 (1). 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 G 1 . WS cells show a mild to strong hypersensitivity to agents that cause DSBs: ionizing radiation and DNA cross-linking reagents, respectively (41). Interestingly, RTS cells are also hypersensitive to ionizing radiation (47), 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 (48). Ku stimulates the WRN 3Ј-to 5Ј-exonuclease and increases the processivity of the enzyme (48 -50). 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 (49,51). Furthermore, aberrant products of NHEJ reactions have been observed in WS cells (22). 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 ␦, 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 ␤, and stimulates strand displacement DNA synthesis (28). Another important BER enzyme, PARP-1, was recently reported to be the most prominent binder to the WRN RQC domain (52). 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 H 2 O 2 and methyl methanesulfonate (52), indicating the SSBs may not be properly processed in WS cells. For example, in yeast the 3Ј to 5Ј Srs2 helicase shuttles SSBs formed during replication into gap-filling repair pathways as opposed to recombinogenic pathways (53). Similarly, incomplete BER intermediates (small gaps) are shuttled to the HR pathway for resolution (54). 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.
Roles in Telomere Maintenance 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 (41), 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 (55). 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) (55)(56)(57). An ALT pathway in S. cerevisiae is dependent on recombination proteins Rad52 and Rad50 and requires Sgs1 (56,58,59). 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 (60). 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 (41). These results are consistent with a possible role for RecQ helicases in repair and processing of telomeric end structures.
Roles in DNA Damage Response 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 (6). In mammalian cells, WRN tyrosine phosphorylation is induced by bleomycin (␥-irradiation mimic) in a manner dependent on the c-Abl kinase DNA damage response pathway (61). 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 Sphase checkpoint protein Nbs1 (62). BLM and WRN have been shown to be phosphorylated in an ATM-and ATR-dependent manner in response to replication fork stalling (2,63). 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 (40). 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 (1). Furthermore, WRN and BLM co-localize with p53 in response to replication fork stalling (64,65). Thus, RecQ helicases likely play an important role in the cellular response to DNA damage, particularly during S-phase.

Conclusions and Future Perspectives
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 (6,30). 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 (65)(66)(67). In addition, recent evidence indicates that WRN may function as a structural scaffold protein in NHEJ (22). 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.
In conclusion, RecQ helicases may coordinate multiple DNA damage response and repair pathways and appear to be particularly important for dealing with damage during S-phase. The RecQ helicase biochemistry indicates that recombination intermediates are preferred substrates, which may be important for roles upstream in repair to prevent inappropriate recombination but also downstream in HR to ensure proper resolution. G-quadruplex DNA is also a preferred substrate, and future work is required to address possible roles for RecQ helicases with these structures in vivo. Presently, we are at a junction where RecQ helicase biochemistry meets human disease, but their precise relationship is not yet understood. Biochemical characterization of other RecQ family members should aid the investigation into the diverse roles of these enzymes in vivo. Defining possible overlapping and distinct roles should undoubtedly provide insight into the molecular pathology of the various human disorders associated with defects in RecQ helicases. Oxidative stress and DNA replication fork collapse can lead to SSBs and/or base modifications, which may be shuttled to an appropriate repair pathway by WRN and possibly BLM (BER and SSB repair (SSBR)). Stalled DNA replication forks and direct DNA damage can lead to DSBs that may be shuttled to the HR pathway, perhaps by BLM, or the NHEJ pathway in which WRN may be involved. BLM and WRN have been shown to resolve recombination intermediates at late stages of HR. Details of these pathways are described in the text.