Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase

There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.     

Structural basis for DNA strand separation by a hexameric replicative helicase

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.     

Human RECQ5beta helicase promotes strand exchange on synthetic DNA structures resembling a stalled replication fork

The role of the human RECQ5β helicase in the maintenance of genomic stability remains elusive. Here we show that RECQ5β promotes strand exchange between arms of synthetic forked DNA structures resembling a stalled replication fork in a reaction dependent on ATP hydrolysis. BLM and WRN can also promote strand exchange on these structures. However, in the presence of human replication protein A (hRPA), the action of these RecQ-type helicases is strongly biased towards unwinding of the parental duplex, an effect not seen with RECQ5β. A domain within the non-conserved portion of RECQ5β is identified as being important for its ability to unwind the lagging-strand arm and to promote strand exchange on hRPA-coated forked structures. We also show that RECQ5β associates with DNA replication factories in S phase nuclei and persists at the sites of stalled replication forks after exposure of cells to UV irradiation. Moreover, RECQ5β is found to physically interact with the polymerase processivity factor proliferating cell nuclear antigen in vitro and in vivo. Collectively, these findings suggest that RECQ5β may promote regression of stalled replication forks to facilitate the bypass of replication-blocking lesions by template-switching. Loss of such activity could explain the elevated level of mitotic crossovers observed in RECQ5β-deficient cells.     

Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl₂. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The K_m values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.     

The T4 phage UvsW protein contains both DNA unwinding and strand annealing activities

UvsW protein belongs to the SF2 helicase family and is one of three helicases found in T4 phage. UvsW governs the transition from origin-dependent to origin-independent replication through the dissociation of R-loops located at the T4 origins of replication. Additionally, in vivo evidence indicates that UvsW plays a role in recombination-dependent replication and/or DNA repair. Here, the biochemical properties of UvsW helicase are described. UvsW is a 3' to 5' helicase that unwinds a wide variety of substrates, including those resembling stalled replication forks and recombination intermediates. UvsW also contains a potent single-strand DNA annealing activity that is enhanced by ATP hydrolysis but does not require it. The annealing activity is inhibited by the non-hydrolysable ATP analog (adenosine 5'-O-(thiotriphosphate)), T4 single-stranded DNA-binding protein (gp32), or a small 8.8-kDa polypeptide (UvsW.1). Fluorescence resonance energy transfer experiments indicate that UvsW and UvsW.1 form a complex, suggesting that the UvsW helicase may exist as a heterodimer in vivo. Fusion of UvsW and UvsW.1 results in a 68-kDa protein having nearly identical properties as the UvsW-UvsW.1 complex, indicating that the binding locus of UvsW.1 is close to the C terminus of UvsW. The biochemical properties of UvsW are similar to the RecQ protein family and suggest that the annealing activity of these helicases may also be modulated by protein-protein interactions. The dual activities of UvsW are well suited for the DNA repair pathways described for leading strand lesion bypass and synthesis-dependent strand annealing.     

Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

Dimerization of simian virus 40 T-antigen hexamers (TAg_H) into double hexamers (TAg_DH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAg_DH with DNA during unwinding, we examined the binding of TAg_DH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAg_DH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAg_DH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ~17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAg_H encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAg_H interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.     

The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA

The product of the gene mutated in Bloom's syndrome, BLM, is a 3′–5′ DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg²⁺, is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.     

Unwinding of the nascent lagging strand by Rep and PriA enables the direct restart of stalled replication forks

During origin-independent replisome assembly, the replication restart protein PriC prefers to load the replication fork helicase, DnaB, to stalled replication forks where there is a gap in the nascent leading strand. However, this activity can be obstructed if the 5'-end of the nascent lagging strand is near the template branch point. Here we provide biochemical evidence that the helicase activities of Rep and PriA function to unwind the nascent lagging strand DNA at such stalled replication forks. PriC then loads the replicative helicase, DnaB, onto the newly generated, single-stranded template for the purposes of replisome assembly and duplex unwinding ahead of the replication fork. Direct rescue of replication forks by the Rep-PriC and PriA-PriC pathways in this manner may contribute to genomic stability by avoiding the potential dangers of fork breakage inherent to recombination-dependent restart pathways.     

Unwinding of forked DNA structures by UvrD

Many studies have demonstrated the need for processing of blocked replication forks to underpin genome duplication. UvrD helicase in Escherichia coli has been implicated in the processing of damaged replication forks, or the recombination intermediates formed from damaged forks. Here we show that UvrD can unwind forked DNA structures, in part due to the ability of UvrD to initiate unwinding from discontinuities within the phosphodiester backbone of DNA. UvrD does therefore have the capacity to target DNA intermediates of replication and recombination. Such an activity resulted in unwinding of what would be the parental duplex DNA ahead of either a stalled replication fork or a D-loop formed by recombination. However, UvrD had a substrate preference for fork structures having a nascent lagging strand at the branch point but no leading strand. Furthermore, at such structures the polarity of UvrD altered so that unwinding of the lagging strand predominated. This reaction is reminiscent of the PriC-Rep pathway of replication restart, suggesting that UvrD and Rep may have at least partially redundant functions.     

DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Replication fork regression in vitro by the Werner syndrome protein (WRN): holliday junction formation, the effect of leading arm structure and a potential role for WRN exonuclease activity

The premature aging and cancer-prone disease Werner syndrome stems from loss of WRN protein function. WRN deficiency causes replication abnormalities, sensitivity to certain genotoxic agents, genomic instability and early replicative senescence in primary fibroblasts. As a RecQ helicase family member, WRN is a DNA-dependent ATPase and unwinding enzyme, but also possesses strand annealing and exonuclease activities. RecQ helicases are postulated to participate in pathways responding to replication blockage, pathways possibly initiated by fork regression. In this study, a series of model replication fork substrates were used to examine the fork regression capability of WRN. Our results demonstrate that WRN catalyzes fork regression and Holliday junction formation. This process is an ATP-dependent reaction that is particularly efficient on forks containing single-stranded gaps of at least 11–13 nt on the leading arm at the fork junction. Importantly, WRN exonuclease activity, by digesting the leading daughter strand, enhances regression of forks with smaller gaps on the leading arm, thus creating an optimal structure for regression. Our results suggest that the multiple activities of WRN cooperate to promote replication fork regression. These findings, along with the established cellular consequences of WRN deficiency, strongly support a role for WRN in regression of blocked replication forks.     

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase

ScPif1 DNA helicase is the prototypical member of a 5′-to-3′ helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5′-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA–RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo.     

PriB stimulates PriA helicase via an interaction with single-stranded DNA

The frequency with which replication forks break down in all organisms requires that specific mechanisms ensure completion of genome duplication. In Escherichia coli a major pathway for reloading of the replicative apparatus at sites of fork breakdown is dependent on PriA helicase. PriA acts in conjunction with PriB and DnaT to effect loading of the replicative helicase DnaB back onto the lagging strand template, either at stalled fork structures or at recombination intermediates. Here we showed that PriB stimulates PriA helicase, acting to increase the apparent processivity of PriA. This stimulation correlates with the ability of PriB to form a ternary complex with PriA and DNA structures containing single-stranded DNA, suggesting that the known single-stranded DNA binding function of PriB facilitates unwinding by PriA helicase. This enhanced apparent processivity of PriA might play an important role in generating single-stranded DNA at stalled replication forks upon which to load DnaB. However, stimulation of PriA by PriB is not DNA structure-specific, demonstrating that targeting of stalled forks and recombination intermediates during replication restart likely resides with PriA alone.     

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.     

Structural and Mechanistic Insight into DNA Unwinding by Deinococcus radiodurans UvrD

DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3′-5′ direction and shows ATP-dependent 3′-5′, and surprisingly also, 5′-3′ helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD’s 3′-5′ and 5′-3′ helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5′-3′ helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo.     

A DNA helicase from human cells.

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.     

Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase

TWINKLE is a nucleus-encoded human mitochondrial (mt)DNA helicase. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. To understand the biochemical basis of these diseases, it is important to define the roles of TWINKLE in mtDNA metabolism by studying its enzymatic activities. To this end, we purified native TWINKLE from Escherichia coli. The recombinant TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. Probing into the DNA unwinding activity, we discovered that the efficiency of unwinding is greatly enhanced in the presence of a heterologous single strand-binding protein or a single-stranded (ss) DNA that is complementary to the unwound strand. We show that TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs that interferes with unwinding in the absence of gp2.5 or ssDNA trap. Furthermore, only ssDNA and not double-stranded (ds)DNA competitively inhibits the annealing activity, although both DNAs bind with high affinities. This implies that dsDNA binds to a site that is distinct from the ssDNA-binding site that promotes annealing. Fluorescence anisotropy competition binding experiments suggest that TWINKLE has more than one ssDNA-binding sites, and we speculate that a surface-exposed ssDNA-specific site is involved in catalyzing DNA annealing. We propose that the strand annealing activity of TWINKLE may play a role in recombination-mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork regression during repair of damaged DNA replication forks.     

Human DNA helicase V, a novel DNA unwinding enzyme from HeLa cells.

Using a strand-displacement assay with 32P labeled oligonucleotide annealed to M13 ssDNA we have purified to apparent homogeneity and characterized a novel DNA unwinding enzyme from HeLa cell nuclei, human DNA helicase V (HDH V). This is present in extremely low abundance in the cells and has the highest turnover rate among other human helicases. From 300 grams of cultured cells only 0.012 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The enzyme also shows ATPase activity dependent on single-stranded DNA and has an apparent molecular weight of 92 kDa by SDS-polyacrylamide gel electrophoresis. Only ATP or dATP hydrolysis supports the unwinding activity. The helicase requires a divalent cation (Mg2+ > Mn2+) at an optimum concentration of 1.0 mM for activity; it unwinds DNA duplexes less than 25 bp long and having a ssDNA stretch as short as 49 nucleotides. A replication fork-like structure is not required to perform DNA unwinding. HDH V cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids; it unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand, a polarity similar to the previously described human DNA helicases I and III (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acid Res. 20, 5329-5337, 1992) and opposite to that of human DNA helicase IV (Tuteja et al. Nucleic Acid Res. 19, 3613-3618, 1991).     

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.