Analysis of helicase activity and substrate specificity of Drosophila RECQ5

RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3′ Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5′ Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM.     

Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1

RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.     

RECQ1 plays a distinct role in cellular response to oxidative DNA damage

RECQ1 is the most abundant RecQ homolog in humans but its functions have remained mostly elusive. Biochemically, RECQ1 displays distinct substrate specificities from WRN and BLM, indicating that these RecQ helicases likely perform non-overlapping functions. Our earlier work demonstrated that RECQ1-deficient cells display spontaneous genomic instability. We have obtained key evidence suggesting a unique role of RECQ1 in repair of oxidative DNA damage. We show that similar to WRN, RECQ1 associates with PARP-1 in nuclear extracts and exhibits direct protein interaction in vitro. Deficiency in WRN or BLM helicases have been shown to result in reduced homologous recombination and hyperactivation of PARP under basal condition. However, RECQ1-deficiency did not lead to PARP activation in undamaged cells and nor did it result in reduction in homologous recombination repair. In stark contrast to what is seen in WRN-deficiency, RECQ1-deficient cells hyperactivate PARP in a specific response to H₂O₂ treatment. RECQ1-deficient cells are more sensitive to oxidative DNA damage and exposure to oxidative stress results in a rapid and reversible recruitment of RECQ1 to chromatin. Chromatin localization of RECQ1 precedes WRN helicase, which has been shown to function in oxidative DNA damage repair. However, oxidative DNA damage-induced chromatin recruitment of these RecQ helicases is independent of PARP activity. As other RecQ helicases are known to interact with PARP-1, this study provides a paradigm to delineate specialized and redundant functions of RecQ homologs in repair of oxidative DNA damage.     

Unique and important consequences of RECQ1 deficiency in mammalian cells

Five members of the RecQ subfamily of DEx-H-containing DNA helicases have been identified in both human and mouse, and mutations in BLM, WRN, and RECQ4 are associated with human diseases of premature aging, cancer, and chromosomal instability. Although a genetic disease has not been linked to RECQ1 mutations, RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage.(1) Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous γ-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks.(2) Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin.(2) RECQ1 catalytic activities(3) and interactions with DNA repair proteins(2,4,5) are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis.     

dRecQ4 Is Required for DNA Synthesis and Essential for Cell Proliferation in Drosophila

Background

The family of RecQ DNA helicases plays an important role in the maintenance of genomic integrity. Mutations in three of the five known RecQ family members in humans, BLM, WRN and RecQ4, lead to disorders that are characterized by predisposition to cancer and premature aging.

Methodology/Principal Findings

To address the in vivo functions of Drosophila RecQ4 (dRecQ4), we generated mutant alleles of dRecQ4 using the targeted gene knock-out technique. Our data show that dRecQ4 mutants are homozygous lethal with defects in DNA replication, cell cycle progression and cell proliferation. Two sets of experiments suggest that dRecQ4 also plays a role in DNA double strand break repair. First, mutant animals exhibit sensitivity to gamma irradiation. Second, the efficiency of DsRed reconstitution via single strand annealing repair is significantly reduced in the dRecQ4 mutant animals. Rescue experiments further show that both the N-terminal domain and the helicase domain are essential to dRecQ4 function in vivo. The N-terminal domain is sufficient for the DNA repair function of dRecQ4.

Conclusions/Significance

Together, our results show that dRecQ4 is an essential gene that plays an important role in not only DNA replication but also DNA repair and cell cycle progression in vivo.     

RECQ1 possesses DNA branch migration activity

RecQ helicases are essential for the maintenance of genome stability. Five members of the RecQ family have been found in humans, including RECQ1, RECQ5, BLM, WRN, and RECQ4; the last three are associated with human diseases. At this time, only BLM and WRN helicases have been extensively characterized, and the information on the other RecQ helicases has only started to emerge. Our current paper is focused on the biochemical properties of human RECQ1 helicase. Recent cellular studies have shown that RECQ1 may participate in DNA repair and homologous recombination, but the exact mechanisms of how RECQ1 performs its cellular functions remain largely unknown. Whereas RECQ1 possesses poor helicase activity, we found here that the enzyme efficiently promotes DNA branch migration. Further analysis revealed that RECQ1 catalyzes unidirectional three-stranded branch migration with a 3' --> 5' polarity. We show that this RECQ1 activity is instrumental in specific disruption of joint molecules (D-loops) formed by a 5' single-stranded DNA invading strand, which may represent dead end intermediates of homologous recombination in vivo. The newly found enzymatic properties of the RECQ1 helicase may have important implications for the function of RECQ1 in maintenance of genomic stability.     

Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G₁, after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.The RecQ helicases are a family of DNA-unwinding enzymes essential for the maintenance of genome integrity in all kingdoms of life. Five RecQ enzymes have been found in human cells: RECQ1 (also called RECQL or RECQL1), BLM (RECQ2 or RECQL3), WRN (RECQ3 or RECQL2), RECQ4 (RECQL4), and RECQ5 (RECQL5) (3, 7). Here we refer to these helicases as RECQ1, RECQ4, and RECQ5, without the “L” that is present in the official gene names. Mutations in the BLM, WRN, and RECQ4 genes are linked to Bloom syndrome (BS), Werner syndrome (WS), and the subset of Rothmund-Thomson syndrome (RTS) patients at high risk of developing osteosarcomas, respectively (19, 31, 71). RECQ4 mutations have also been associated with RAPADILINO and Baller-Gerold syndrome (56, 61). Although these disorders are all associated with inherent genomic instability and cancer predisposition, they show distinct clinical features, suggesting that BLM, WRN, and RECQ4 are involved in different aspects of DNA metabolism. However, the molecular events underlying the pathogenesis of BS, WS, and RTS remain obscure. Mutations in the remaining two human RecQ helicase genes, RECQ1 and RECQ5, have not as yet been identified as causes of either genomic instability or heritable cancer predisposition disorders.Several lines of evidence suggest that RecQ helicases play an important role in DNA replication control (3, 10). In particular, RecQ helicases are thought to facilitate replication by preserving the integrity of stalled replication forks and by remodeling or repairing damaged or collapsed forks to allow the resumption of replication. Consistent with these ideas, several investigators have shown that primary fibroblasts from BS, WS, and RTS patients and RecQ5-deficient mouse embryonic fibroblasts all show differential hypersensitivity to agents that perturb DNA replication (12, 14, 26, 29). Moreover, BLM and WRN are recruited to DNA replication forks after replicative stress, and DNA fiber track analyses have shown that both BLM and WRN are required for normal fork progression after DNA damage or replication arrest (11-13, 47, 54). In particular, BLM in conjunction with DNA topoisomerase III and two other accessory proteins, RMI-1 and RMI-2, has been shown to catalyze the resolution of double-Holliday-junction recombination intermediates to generate noncrossover products. This dissolution reaction could play an important role in the error-free recombinational repair of damaged or stalled forks during S phase (57, 67). WRN also appears to promote error-free repair by contributing to the resolution of gene conversion events to generate noncrossover products (46). In line with the above observations, WRN and BLM can be found associated with replication foci or other DNA damage response proteins in damaged cells. In contrast, in unperturbed cells, a majority of each protein is found in the nucleolus (WRN) or associated with PML bodies (BLM) (5, 37, 62).RECQ4 has also been implicated in DNA replication. Recent studies have shown that hypomorphic mutants of the Drosophila melanogaster homolog of human RECQ4, DmRECQ4, have reduced DNA replication-dependent chorion gene amplification (65). These findings are thus consistent with a postulated role for Xenopus laevis RECQ4 (XRECQ4) in the initiation of DNA replication (39, 48). The N terminus of XRECQ4 bears homology to the N termini of the yeast proteins Sld2 (Saccharomyces cerevisiae [budding yeast]) and DRC1 (Schizosaccharomyces pombe [fission yeast]), which play a central role, in association with budding yeast Dpb11 and the fission yeast homolog Cut5/Rad4, in the establishment of DNA replication forks (38, 41, 63). Consistently, the N terminus of XRECQ4 has been shown to interact with the X. laevis variant of Cut5, and XRECQ4 depletion severely perturbs DNA replication initiation in X. laevis egg extracts (39, 48). The notion that the function of XRECQ4 is evolutionarily conserved in mammals is supported by the observations that the human protein can complement its Xenopus counterpart in cell-free assays for replication initiation and that depletion of human RECQ4 inhibits cellular proliferation and DNA synthesis (39, 48). Moreover, deletion of the N-terminal region of mouse RECQ4 has been shown to be an embryonic lethal mutation (27). These observations suggest that vertebrate RECQ4 might be a functional homolog of Sld2/DRC11, although its precise function during replication initiation and progression is not known. Recent results, published while this work was in progress, indicate that human RECQ4 interacts with the MCM replicative complex during replication initiation and that this interaction is regulated by CDK phosphorylation of RECQ4 (69). These findings, together with our results below, provide clues to the mechanism regulating RECQ4 interaction with the replication machinery.RECQ1 is the most abundant of the human RecQ helicases and was the first of the human RecQ proteins to be discovered on the basis of its potent ATPase activity (50). Despite this, little is known about the cellular functions of RECQ1, and no human disease associations have been identified to date. Recent studies have shown that RECQ1 is involved in the maintenance of genome integrity and that RECQ1 depletion affects cellular proliferation (51). Moreover, biochemical studies have shown that RECQ1 and BLM display distinct substrate specificities, indicating that these helicases are likely to perform nonoverlapping functions (43). These results suggest an important—though as yet mechanistically ill-defined—role for RECQ1 in cell cycle progression and/or DNA repair (52).In order to better delineate the role of human RecQ helicases in DNA replication, we investigated the in vivo interactions of all five human RecQ enzymes with three well-characterized human DNA replication origins in quantitative chromatin immunoprecipitation (ChIP) assays. We also determined how nascent-origin-dependent DNA synthesis, chromatin binding of replication proteins, origin firing frequency, and replication fork rates were altered by depleting specific human RecQ helicase proteins. We found that only two of the five human RecQ helicases, RECQ1 and RECQ4, specifically interact with origins in unperturbed cells. Our results provide new mechanistic insight into the distinct roles of human RECQ1 and RECQ4 in DNA replication initiation and in replication fork progression.     

RECQ1 helicase interacts with human mismatch repair factors that regulate genetic recombination

Understanding the molecular and cellular functions of RecQ helicases has attracted considerable interest since several human diseases characterized by premature aging and/or cancer have been genetically linked to mutations in genes of the RecQ family. Although a human disease has not yet been genetically linked to a mutation in RECQ1, the prominent roles of RecQ helicases in the maintenance of genome stability suggest that RECQ1 helicase is likely to be important in vivo.To acquire a better understanding of RECQ1 cellular and molecular functions, we have investigated its protein interactions. Using a co-immunoprecipitation approach, we have identified several DNA repair factors that are associated with RECQ1 in vivo. Direct physical interaction of these repair factors with RECQ1 was confirmed with purified recombinant proteins. Importantly, RECQ1 stimulates the incision activity of human exonuclease 1 and the mismatch repair recognition complex MSH2/6 stimulates RECQ1 helicase activity. These protein interactions suggest a role of RECQ1 in a pathway involving mismatch repair factors. Regulation of genetic recombination, a proposed role for RecQ helicases, is supported by the identified RECQ1 protein interactions and is discussed.     

Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis

RecQ helicases are critical for the maintenance of genomic stability. The Arabidopsis RecQ helicase RECQ4A is the functional counterpart of human BLM, which is mutated in the genetic disorder Bloom’s syndrome. RECQ4A performs critical roles in regulation of homologous recombination (HR) and DNA repair. Loss of RECQ4A leads to elevated HR frequencies and hypersensitivity to genotoxic agents. Through complementation studies, we were now able to demonstrate that the N-terminal region and the helicase activity of RECQ4A are both essential for the cellular response to replicative stress induced by methyl methanesulfonate and cisplatin. In contrast, loss of helicase activity or deletion of the N-terminus only partially complemented the mutant hyper-recombination phenotype. Furthermore, the helicase-deficient protein lacking its N-terminus did not complement the hyper-recombination phenotype at all. Therefore, RECQ4A seems to possess at least two different and independent sub-functions involved in the suppression of HR. By in vitro analysis, we showed that the helicase core was able to regress an artificial replication fork. Swapping of the terminal regions of RECQ4A with the closely related but functionally distinct helicase RECQ4B indicated that in contrast to the C-terminus, the N-terminus of RECQ4A was required for its specific functions in DNA repair and recombination.     

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers

Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.     

Biochemical Characterization of AtRECQ3 Reveals Significant Differences Relative to Other RecQ Helicases

Members of the conserved RecQ helicase family are important for the preservation of genomic stability. Multiple RecQ homologs within one organism raise the question of functional specialization. Whereas five different homologs are present in humans, the model plant Arabidopsis (Arabidopsis thaliana) carries seven RecQ homologs in its genome. We performed biochemical analysis of AtRECQ3, expanded upon a previous analysis of AtRECQ2, and compared their properties. Both proteins differ in their domain composition. Our analysis demonstrates that they are 3′ to 5′ helicases with similar activities on partial duplex DNA. However, they promote different outcomes with synthetic DNA structures that mimic Holliday junctions or a replication fork. AtRECQ2 catalyzes Holliday junction branch migration and replication fork regression, while AtRECQ3 cannot act on intact Holliday junctions. The observed reaction of AtRECQ3 on the replication fork is in line with unwinding the lagging strand. On nicked Holliday junctions, which have not been intensively studied with RecQ helicases before, AtRECQ3, but not AtRECQ2, shows a clear preference for one unwinding mechanism. In addition, AtRECQ3 is much more efficient at catalyzing DNA strand annealing. Thus, AtRECQ2 and AtRECQ3 are likely to perform different tasks in the cell, and AtRECQ3 differs in its biochemical properties from all other eukaryotic RECQ helicases characterized so far.RecQ helicases are important players in the maintenance of genomic stability in prokaryotes and eukaryotes (for reviews covering several aspects of RecQ helicases, see Bachrati and Hickson, 2003; Hickson, 2003; Opresko et al., 2004; Sharma et al., 2006; Brosh and Bohr, 2007; Hanada and Hickson, 2007; Bachrati and Hickson, 2008). Interestingly, organisms contain different numbers of RecQ homologs. In humans, mutations in three of the five RecQ helicases have been linked to distinct, cancer-associated genetic diseases. Consequently, the human RecQ helicases have been intensively studied. All RecQ helicases share a characteristic helicase domain, due to which they are classified as RecQ helicases. In addition, a RecQ C-terminal (RQCt) and/or a Helicase and RNAseD C-terminal (HRDC) domain can be identified in some RecQ helicases. The possible functions of the latter in protein stability and DNA and protein binding are summarized, for example, in a recent review (Chu and Hickson, 2009). From the enzymatic point of view, most RecQ helicases both unwind DNA and promote the opposite reaction, called strand annealing. In addition, some RecQ homologs combine these activities to catalyze branch migration.One of the central questions regarding RecQ helicases is the extent to which the homologs have unique or overlapping functions (Ellis et al., 2008). This question originally raised for the human RecQ helicases is of equal importance for other organisms, such as plants. Analysis of plant RecQ homologs could reveal species- or kingdom-specific tasks, and such comparisons could shed light on evolutionary questions, such as how selection pressures induce multiple homologs within a single species.In the dicotyledonous model plant Arabidopsis (Arabidopsis thaliana), seven RecQ helicase homologs have been identified (Hartung et al., 2000; Hartung and Puchta, 2006). A biochemical comparative analysis may provide information about the molecular basis of their cellular functions, which are likely to be different. As has been shown, sequence homology does not necessarily imply functional homology (Hartung et al., 2007).Previously, we were able to characterize basic biochemical properties of the helicase RECQ2 of the model plant Arabidopsis (Kobbe et al., 2008). In this study, we chose to study AtRECQ3 as a RecQ helicase that differs in the domain structure from AtRECQ2, lacking the HRDC domain and the winged helix subdomain of the RQCt domain (Fig. 1A). AtRECQ3 has the same domain structure as human RECQ5β. AtRECQ3 was expressed in Escherichia coli, purified, and biochemically characterized. The specificity of AtRECQ2 and AtRECQ3 was compared with several substrates, extending the analyzed spectrum of substrates for AtRECQ2 and for RecQ helicases in general. We identified several differences in the enzymatic properties of AtRECQ2 and AtRECQ3, suggesting that these two enzymes might play different roles in vivo.Open in a separate windowFigure 1.Domain structure, purification, and directionality of AtRECQ2 and AtRECQ3. A, Schematic drawing of the domain structure of AtRECQ2 and AtRECQ3. In the helicase domain, the different helicase motifs (0, I, Ia, II, III, IV, V, and VI) are depicted in dark gray. B, 10% SDS-PAGE analysis stained with colloidal Coomassie Brilliant Blue of representative purifications of AtRECQ2-K117M (lane 1, 0.5 pmol), AtRECQ2 (lane 2, 0.8 pmol), AtRECQ3-K64M (lane 3, 0.9 pmol), and AtRECQ3 (lane 4, 1.25 pmol). The proteins were overexpressed in E. coli and purified by nickel-immobilized metal ion affinity chromatography and Calmodulin affinity chromatography. The gray arrow indicates AtRECQ2/-K117M with a predicted molecular mass of 85.5 kD, and the black arrow indicates AtRECQ3/-K64M with a predicted molecular mass of 86 kD. C, AtRECQ2 (8 nm), AtRECQ3 (8 nm), AtRECQ3-K64M (8 nm), and AtRECQ2-K117M (5 nm) were incubated with 150 pm of the indicated substrates for 30 min at 37°C. Error bars indicate sd of the mean of three independent experiments.     

AtRECQ2, a RecQ helicase homologue from Arabidopsis thaliana, is able to disrupt various recombinogenic DNA structures in vitro

RecQ helicases play an important role in the maintenance of genomic stability in pro- and eukaryotes. This is highlighted by the human genetic diseases Werner, Bloom's and Rothmund–Thomson syndrome, caused by respective mutations in three of the five human RECQ genes. The highest numbers of RECQ homologous genes are found in plants, e.g. seven in Arabidopsis thaliana . However, only limited information is available on the functions of plant RecQ helicases, and no biochemical characterization has been performed. Here, we demonstrate that AtRECQ2 is a (d)NTP-dependent 3'→5' DNA helicase. We further characterized its basal properties and its action on various partial DNA duplexes. Importantly, we demonstrate that AtRECQ2 is able to disrupt recombinogenic structures: by disrupting various D-loop structures, AtRECQ2 may prevent non-productive recombination events on the one hand, and may channel repair processes into non-recombinogenic pathways on the other hand, thus facilitating genomic stability. We show that a synthetic partially mobile Holliday junction is processed towards splayed-arm products, possibly indicating a branch migration function for AtRECQ2. The biochemical properties defined in this work support the hypothesis that AtRECQ2 might be functionally orthologous to the helicase part of the human RecQ homologue HsWRN.     

MCM10 mediates RECQ4 association with MCM2‐7 helicase complex during DNA replication

Mutations in RECQ4, a member of the RecQ family of DNA helicases, have been linked to the progeroid disease Rothmund–Thomson Syndrome. Attempts to understand the complex phenotypes observed in recq4‐deficient cells suggest a potential involvement in DNA repair and replication, yet the molecular basis of the function of RECQ4 in these processes remains unknown. Here, we report the identification of a highly purified chromatin‐bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2‐7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Importantly, complex formation and the association of RECQ4 with the replication origin are cell‐cycle regulated. Furthermore, we show that MCM10 is essential for the integrity of the RECQ4–MCM replicative helicase complex. MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity, and that this interaction may be modulated by cyclin‐dependent kinase phosphorylation. Thus, these studies show that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells.     

Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A

The RecQ helicases are involved in several aspects of DNA metabolism. Five members of the RecQ family have been found in humans, but only two of them have been carefully characterized, BLM and WRN. In this work, we describe the enzymatic characterization of RECQ1. The helicase has 3' to 5' polarity, cannot start the unwinding from a blunt-ended terminus, and needs a 3'-single-stranded DNA tail longer than 10 nucleotides to open the substrate. However, it was also able to unwind a blunt-ended duplex DNA with a "bubble" of 25 nucleotides in the middle, as previously observed for WRN and BLM. We show that only short DNA duplexes (<30 bp) can be unwound by RECQ1 alone, but the addition of human replication protein A (hRPA) increases the processivity of the enzyme (>100 bp). Our studies done with Escherichia coli single-strand binding protein (SSB) indicate that the helicase activity of RECQ1 is specifically stimulated by hRPA. This finding suggests that RECQ1 and hRPA may interact also in vivo and function together in DNA metabolism. Comparison of the present results with previous studies on WRN and BLM provides novel insight into the role of the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA.     

The Human RecQ helicases, BLM and RECQ1, display distinct DNA substrate specificities

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3'-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.     

A prominent β-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation

RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1(49-616)) shows a prominent β-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the β-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the β-hairpin is required for dimer formation in RECQ1(49-616) and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1(49-616) dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein-protein contacts are required for tetramer formation, one involves the β-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.     

Comparison of proliferation and genomic instability responses to WRN silencing in hematopoietic HL60 and TK6 cells

Background

Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis.

Methodology/Principal Findings

We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges.

Conclusions/Significance

Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency.     

Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding

The Drosophila melanogaster RECQ5/QE gene encodes a member of the DNA helicase family comprising the Escherichia coli RecQ protein and products of the human Bloom’s, Werner’s, and Rothmund-Thomson syndrome genes. The full-length product of RECQ5/QE was expressed in the baculovirus system and was purified. Gel filtration experiments indicated that RECQ5/QE was present in an oligomeric state. The RECQ5/QE protein hydrolyzed ATP and even more actively GTP in the presence of single-stranded DNA. ATP drove the DNA helicase activity of RECQ5/QE, whereas GTP had little effect. GTP exhibited a stimulatory effect on DNA unwinding when it was used together with ATP. This effect was more apparent with non-hydrolyzable GTP analogs, such as GTPγS and GMPPNP. These results indicate that GTP binding to RECQ5/QE triggers its DNA helicase activity. GTP binding increased the rate of strand separation without affecting the S_0.5 (K_m) values for the substrates during the DNA helicase reaction. The data collectively suggest that the RECQ5/QE protein is activated upon GTP binding through the ATP-binding site.     

The zinc-binding motif of human RECQ5beta suppresses the intrinsic strand-annealing activity of its DExH helicase domain and is essential for the helicase activity of the enzyme

RecQ family helicases, functioning as caretakers of genomic integrity, contain a zinc-binding motif which is highly conserved among these helicases, but does not have a substantial structural similarity with any other known zinc-finger folds. In the present study, we show that a truncated variant of the human RECQ5beta helicase comprised of the conserved helicase domain only, a splice variant named RECQ5alpha, possesses neither ATPase nor DNA-unwinding activities, but surprisingly displays a strong strand-annealing activity. In contrast, fragments of RECQ5beta including the intact zinc-binding motif, which is located immediately downstream of the helicase domain, exhibit much reduced strand-annealing activity but are proficient in DNA unwinding. Quantitative measurements indicate that the regulatory role of the zinc-binding motif is achieved by enhancing the DNA-binding affinity of the enzyme. The novel intramolecular modulation of RECQ5beta catalytic activity mediated by the zinc-binding motif may represent a universal regulation mode for all RecQ family helicases.     

DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion

DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions.