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.     

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.     

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 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.     

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.     

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.     

RECQ1 is required for cellular resistance to replication stress and catalyzes strand exchange on stalled replication fork structures

RECQ1 is the most abundant of the five human RecQ helicases, but little is known about its biological significance. Recent studies indicate that RECQ1 is associated with origins of replication, suggesting a possible role in DNA replication. However, the functional role of RECQ1 at damaged or stalled replication forks is still unknown. Here, for the first time, we show that RECQ1 promotes strand exchange on synthetic stalled replication fork-mimicking structures and comparatively analyze RECQ1 with the other human RecQ helicases. RECQ1 actively unwinds the leading strand of the fork, similar to WRN, while RECQ4 and RECQ5β can only unwind the lagging strand of the replication fork. Human replication protein A modulates the strand exchange activity of RECQ1 and shifts the equilibrium more to the unwinding mode, an effect also observed for WRN. Stable depletion of RECQ1 affects cell proliferation and renders human cells sensitive to various DNA damaging agents that directly or indirectly block DNA replication fork progression. Consequently, loss of RECQ1 activates DNA damage response signaling, leads to hyper-phosphorylation of RPA32 and activation of CHK1, indicating replication stress. Furthermore, depletion of RECQ1 leads to chromosomal condensation defects and accumulation of under-condensed chromosomes. Collectively, our observations provide a new insight into the role of RECQ1 in replication fork stabilization and its role in the DNA damage response to maintain genomic stability.     

RECQ1 is required for cellular resistance to replication stress and catalyzes strand exchange on stalled replication fork structures

RECQ1 is the most abundant of the five human RecQ helicases, but little is known about its biological significance. Recent studies indicate that RECQ1 is associated with origins of replication, suggesting a possible role in DNA replication. However, the functional role of RECQ1 at damaged or stalled replication forks is still unknown. Here, for the first time, we show that RECQ1 promotes strand exchange on synthetic stalled replication fork-mimicking structures and comparatively analyze RECQ1 with the other human RecQ helicases. RECQ1 actively unwinds the leading strand of the fork, similar to WRN, while RECQ4 and RECQ5β can only unwind the lagging strand of the replication fork. Human replication protein A modulates the strand exchange activity of RECQ1 and shifts the equilibrium more to the unwinding mode, an effect also observed for WRN. Stable depletion of RECQ1 affects cell proliferation and renders human cells sensitive to various DNA damaging agents that directly or indirectly block DNA replication fork progression. Consequently, loss of RECQ1 activates DNA damage response signaling, leads to hyper-phosphorylation of RPA32 and activation of CHK1, indicating replication stress. Furthermore, depletion of RECQ1 leads to chromosomal condensation defects and accumulation of under-condensed chromosomes. Collectively, our observations provide a new insight into the role of RECQ1 in replication fork stabilization and its role in the DNA damage response to maintain genomic stability.     

Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide

Proteins belonging to the highly conserved RecQ helicase family are essential for the maintenance of genomic stability. Here, we describe the biochemical properties of the human RECQ5beta protein. Like BLM and WRN, RECQ5beta is an ATP-dependent 3'-5' DNA helicase that can promote migration of Holliday junctions. However, RECQ5beta required the single-stranded DNA-binding protein RPA in order to mediate the efficient unwinding of oligonucleotide-based substrates. Surprisingly, we found that RECQ5beta possesses an intrinsic DNA strand-annealing activity that is inhibited by RPA. Analysis of deletion variants of RECQ5beta revealed that the DNA helicase activity resides in the conserved N-terminal portion of the protein, whereas strand annealing is mediated by the unique C-terminal domain. Moreover, the strand-annealing activity of RECQ5beta was strongly inhibited by ATPgammaS, a poorly hydrolyzable analog of ATP. This effect was alleviated by mutations in the ATP-binding motif of RECQ5beta, indicating that the ATP-bound form of the protein cannot promote strand annealing. This is the first demonstration of a DNA helicase with an intrinsic DNA strand-annealing function residing in a separate domain.     

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.     

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.     

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.     

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.     

The RecQ helicase AtRECQ4A is required to remove inter-chromosomal telomeric connections that arise during meiotic recombination in Arabidopsis

RecQ helicases are a conserved group of proteins with a role in the maintenance of genome integrity. In Saccharomyces cerevisiae (budding yeast), meiotic recombination is increased in the absence of the RecQ helicase Sgs1. Here we investigated the potential meiotic role of the Sgs1 homologue AtRECQ4A and the closely related AtRECQ4B. Both proteins have been shown to function during recombination in somatic cells, but so far their meiotic role has not been investigated. Both AtRECQ4A and AtRECQ4B were expressed in reproductive tissues. Although immunolocalization studies showed that AtRECQ4A associates with recombination intermediates, we found no evidence that its loss or that of AtRECQ4B had a significant effect on meiotic cross-overs, suggesting functional redundancy with other RECQ family members. Nevertheless, pollen viability decreased in Atrecq4A, resulting in a reduction in fertility, although this was not the case in Atrecq4B. Cytological analysis revealed chromatin bridges between the telomeres of non-homologous chromosomes in Atrecq4A at metaphase I, in some instances accompanied by chromosome fragmentation at anaphase I. The bridges required telomeric repeats and were dependent on meiotic recombination. Immunolocalization confirmed the association of AtRECQ4A with the telomeres during prophase I, which we propose enables dissolution of recombination-dependent telomeric associations. Thus, this study has identified a hitherto unknown role for a member of the RECQ helicase family during meiosis that contributes to the maintenance of chromosome integrity. As telomere structure is generally conserved, it seems likely that these associations may arise during meiosis in other species, where they must also be removed.     

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.     

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.     

Dual DNA unwinding activities of the Rothmund–Thomson syndrome protein,RECQ4

Human RECQ helicases have been linked to distinct clinical diseases with increased cancer rates and premature ageing. All RECQ proteins, except RECQ4, have been shown to be functional helicases. Mutations in RECQ4 lead to Rothmund–Thomson syndrome (RTS), and mouse models reveal that the conserved helicase motifs are required for avoidance of RTS. Furthermore, the amino (N) terminus of RECQ4 shares homology with yeast DNA replication initiation factor, Sld2, and is vital for embryonic development. Here, in contrast to previous reports, we show that RECQ4 exhibits DNA helicase activity. Importantly, two distinct regions of the protein, the conserved helicase motifs and the Sld2‐like N‐terminal domain, each independently promote ATP‐dependent DNA unwinding. Taken together, our data provide the first biochemical clues underlying the molecular function of RECQ4 in DNA replication and genome maintenance.     

Human premature aging, DNA repair and RecQ helicases

Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.     

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.