Replication fork collapse at a protein‐DNA roadblock leads to fork reversal,promoted by the RecQ helicase

Proteins that bind DNA are the cause of the majority of impediments to replication fork progression and can lead to subsequent collapse of the replication fork. Failure to deal with fork collapse efficiently leads to mutation or cell death. Several models have been proposed for how a cell processes a stalled or collapsed replication fork; eukaryotes and bacteria are not dissimilar in terms of the general pathways undertaken to deal with these events. This study shows that replication fork regression, the combination of replication fork reversal leading to formation of a Holliday Junction along with exonuclease digestion, is the preferred pathway for dealing with a collapsed fork in Escherichia coli. Direct endo‐nuclease activity at the replication fork was not observed. The protein that had the greatest effect on these fork processing events was the RecQ helicase, while RecG and RuvABC, which have previously been implicated in this process, were found to play a lesser role. Eukaryotic RecQ homologues, BLM and WRN, have also been implicated in processing events following replication fork collapse and may reflect a conserved mechanism. Finally, the SOS response was not induced by the protein‐DNA roadblock under these conditions, so did not affect fork processing.     

RecQ helicases: guardian angels of the DNA replication fork

Since the original observations made in James German’s Laboratory that Bloom’s syndrome cells lacking BLM exhibit a decreased rate of both DNA chain elongation and maturation of replication intermediates, a large body of evidence has supported the idea that BLM, and other members of the RecQ helicase family to which BLM belongs, play important roles in DNA replication. More recent evidence indicates roles for RecQ helicases in what can broadly be defined as replication fork ‘repair’ processes when, for example, forks encounter lesions or adducts in the template, or when forks stall due to lack of nucleotide precursors. More specifically, several roles in repair of damaged forks via homologous recombination pathways have been proposed. RecQ helicases are generally only recruited to sites of DNA replication following fork stalling or disruption, and they do so in a checkpoint-dependent manner. There, in addition to repair functions, they aid the stabilisation of stalled replication complexes and seem to contribute to the generation and/or transduction of signals that enforce S-phase checkpoints. RecQ helicases also interact physically and functionally with several key players in DNA replication, including RPA, PCNA, FEN1 and DNA polymerase δ. In this paper, we review the evidence that RecQ helicases contribute to the impressively high level of fidelity with which genome duplication is effected.     

The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability

Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges. Together, our results suggest that BLM and RECQL4 have coordinated activities that promote genome stability.     

The Werner and Bloom syndrome proteins help resolve replication blockage by converting (regressed) holliday junctions to functional replication forks

Cells cope with blockage of replication fork progression in a manner that allows DNA synthesis to be completed and genomic instability minimized. Models for resolution of blocked replication involve fork regression to form Holliday junction structures. The human RecQ helicases WRN and BLM (deficient in Werner and Bloom syndromes, respectively) are critical for maintaining genomic stability and thought to function in accurate resolution of replication blockage. Consistent with this notion, WRN and BLM localize to sites of blocked replication after certain DNA-damaging treatments and exhibit enhanced activity on replication and recombination intermediates. Here we examine the actions of WRN and BLM on a special Holliday junction substrate reflective of a regressed replication fork. Our results demonstrate that, in reactions requiring ATP hydrolysis, both WRN and BLM convert this Holliday junction substrate primarily to a four-stranded replication fork structure, suggesting they target the Holliday junction to initiate branch migration. In agreement, the Holliday junction binding protein RuvA inhibits the WRN- and BLM-mediated conversion reactions. Importantly, this conversion product is suitable for replication with its leading daughter strand readily extended by DNA polymerases. Furthermore, binding to and conversion of this Holliday junction are optimal at low MgCl(2) concentrations, suggesting that WRN and BLM preferentially act on the square planar (open) conformation of Holliday junctions. Our findings suggest that, subsequent to fork regression events, WRN and/or BLM could re-establish functional replication forks to help overcome fork blockage. Such a function is highly consistent with phenotypes associated with WRN- and BLM-deficient cells.     

The Werner and Bloom syndrome proteins catalyze regression of a model replication fork

The premature aging and cancer-prone diseases Werner and Bloom syndromes are caused by loss of function of WRN and BLM proteins, respectively. At the cellular level, WRN or BLM deficiency causes replication abnormalities, DNA damage hypersensitivity, and genome instability, suggesting that these proteins might participate in resolution of replication blockage. Although WRN and BLM are helicases belonging to the RecQ family, both have been recently shown to also facilitate pairing of complementary DNA strands. In this study, we demonstrate that both WRN and BLM (but not other selected helicases) can coordinate their unwinding and pairing activities to regress a model replication fork substrate. Notably, fork regression is widely believed to be the initial step in responding to replication blockage. Our findings suggest that WRN and/or BLM might regress replication forks in vivo as part of a genome maintenance pathway, consistent with the phenotypes of WRN- and BLM-deficient cells.     

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.     

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.     

Binding and melting of D-loops by the Bloom syndrome helicase

Bloom syndrome is a rare autosomal disorder characterized by predisposition to cancer and genomic instability. BLM, the structural gene mutated in individuals with the disorder, encodes a DNA helicase belonging to the RecQ family of helicases. These helicases have been established to serve roles in both promoting and preventing recombination. Mounting evidence has implicated a function for BLM during DNA replication; specifically, BLM might be involved in rescuing stalled or collapsed replication forks by a recombination-based mechanism. We have tested this idea by examining the binding and melting activity of BLM on oligonucleotide substrates containing D-loops, DNA structures that model the presumed initial intermediate formed during homologous recombination. We find that BLM preferentially melts those D-loops that are formed more favorably by the strand exchange protein Rad51, but whose polarity could be less favorable for enabling restoration of an active replication fork. We propose a model in which BLM selectively dissociates recombination intermediates likely to be unfavorable for recombination-promoted replication.     

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.     

Functions of RecQ family helicases: possible involvement of Bloom's and Werner's syndrome gene products in guarding genome integrity during DNA replication

Escherichia coli RecQ helicase is a component of the RecF pathway of recombination whose components are required to reassemble a replisome complex at the site of the replication fork after the removal of a lesion. There are at least five RecQ homologues in human cells, including BLM and WRN. The genes encoding BLM and WRN are mutated in the cancer-prone disorder Bloom's syndrome (BS) and the plogeroid disorder Werner's syndrome (WS), respectively. These syndromes are characterized by a high degree of genomic instability, including chromosomal breaks, multiple large deletions, and translocations, and cells derived from BS and WS patients show defects in DNA replication. Recently, it has become clear that a Holliday junction-like structure is formed at stalled replication forks to result in the formation of double-stranded breaks, and recombination plays an important role in the repair of stalled or broken replication forks, leading to the reinitiation of replication. Defects in the processing of stalled replication forks could lead to aberrant recombination events resulting in genetic instability. Recent studies on BLM, WRN, and the RecQ homologue of Saccharomyces cerevisiae, Sgs1, indicate that these RecQ homologues interact with proteins involved in DNA replication, and function in a pathway from the DNA replication check point to homologous recombination.     

RecQ and RecG helicases have distinct roles in maintaining the stability of polypurine.polypyrimidine sequences

DNA triplex structures can block the replication fork and result in double-stranded DNA breaks (DSBs). RecQ and RecG helicases may be important for replication of such sequences as RecQ resolves synthetic triplex DNA structures and RecG mediates replication restart by fork regression. Primer extension on an 88bp triplex-forming polypurine.polypyrimidine (Pu.Py) tract from the PKD1 gene demonstrated that RecQ, but not RecG, facilitated primer extension by T7 DNA polymerase. A high-throughput, dual plasmid screening system using isogenic bacterial lines deficient in RecG, RecQ, or both, revealed that RecQ deficiency increased mutation to sequence flanking this 88bp tract by eight to ten-fold. Although RecG facilitated small deletions in an 88bp mirror repeat-containing sequence, it was absolutely required to maintain a 2.5kb Pu.Py tract containing multiple mirror repeats. These results support a two-tiered model where RecQ facilitates fork progression through triplex-forming tracts and, failing processivity, RecG is critical for replication fork restart.     

A positive involvement of RecQL4 in UV-induced S-phase arrest

RecQL4 belongs to a family of conserved RECQ helicases that are important in maintaining chromosomal integrity. Human patients lacking RecQL4 showed extreme sensitivity to UV and oxidation damage, suggesting that RecQL4 is involved in the damage signaling and/or repair. Here we show that human mutant cells lacking RecQL4 were defective in UV-induced S-phase arrest, whereas cells defective in bloom syndrome protein (BLM), another member of RecQ family exhibited a normal S-phase arrest following UV irradiation. In keeping with this, a targeted inhibition of RecQL4 expression in human 293 cells showed a defect in inducing S-phase (replication) arrest following UV treatment. Human mutant cells lacking RecQL4 protein were also defective in inducing S-phase arrest following hydroxyurea treatment. Together, our results suggest that RecQL4 may have a unique role in replication fork arrest, which may not be shared with other members of RecQ family such as BLM.     

RecQ helicases and cellular responses to DNA damage

The faithful replication of the genome is essential for the survival of all organisms. It is not surprising therefore that numerous mechanisms have evolved to ensure that duplication of the genome occurs with only minimal risk of mutation induction. One mechanism of genome destabilization is replication fork demise, which can occur when a translocating fork meets a lesion or adduct in the template. Indeed, the collapse of replication forks has been suggested to occur in every replicative cell cycle making this a potentially significant problem for all proliferating cells. The RecQ helicases, which are essential for the maintenance of genome stability, are thought to function during DNA replication. In particular, RecQ helicase mutants display replication defects and have phenotypes consistent with an inability to efficiently reinitiate replication following replication fork demise. Here, we review some current models for how replication fork repair might be effected, and discuss potential roles for RecQ helicases in this process.     

RMI1 promotes DNA replication fork progression and recovery from replication fork stress

RMI1 is a member of an evolutionarily conserved complex composed of BLM and topoisomerase IIIα (TopoIIIα). This complex exhibits strand passage activity in vitro, which is likely important for DNA repair and DNA replication in vivo. The inactivation of RMI1 causes genome instability, including elevated levels of sister chromatid exchange and accelerated tumorigenesis. Using molecular combing to analyze DNA replication at the single-molecule level, we show that RMI1 is required to promote normal replication fork progression. The fork progression defect in RMI1-depleted cells is alleviated in cells lacking BLM, indicating that RMI1 functions downstream of BLM in promoting replication elongation. RMI1 localizes to subnuclear foci with BLM and TopoIIIα in response to replication stress. The proper localization of the complex requires a BLM-TopoIIIα-RMI1 interaction and is essential for RMI1 to promote recovery from replication stress. These findings reveal direct roles of RMI1 in DNA replication and the replication stress response, which could explain the molecular basis for its involvement in suppressing sister chromatid exchange and tumorigenesis.     

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.     

Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.     

G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition

To understand the specific genetic instabilities associated with deficiencies in RecQ family helicases, we have studied the substrate preferences of two closely related members of this family, human BLM and Saccharomyces cerevisiae Sgs1p. Here we show that both BLM and Sgs1p preferentially unwind G4 DNA relative to Holliday junction substrates, and that substrate preference reflects binding affinity and maps to the conserved central helicase domain. We identify the porphyrin N-methyl mesoporphyrin IX (NMM) as a specific inhibitor of G4 DNA unwinding, and show that in the presence of NMM the helicase becomes trapped on the NMM–G4 DNA complex, consuming ATP but unable to unwind or dissociate. These results suggest that BLM and Sgs1p function proactively in replication to remove G4 DNA structures which would otherwise present obstacles to fork progression, rather than by promoting recombination to restart a fork that has stalled.     

Delineation of WRN helicase function with EXO1 in the replicational stress response

The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50-Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS). WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkylated base damage. WRN and yEXO1 are associated with each other in vivo. Purified WRN stimulates hEXO1 nuclease activity on DNA substrates associated with a stalled or regressed replication fork. We propose WRN helicase operates in an EXO1-dependent pathway to help cells survive replicational stress. In contrast to WRN, BLM helicase defective in Bloom's syndrome failed to rescue rad50 MMS sensitivity, but partially restored IR resistance, suggesting a delineation of function by the human RecQ helicases.     

RECQL5 and BLM exhibit divergent functions in cells defective for the Fanconi anemia pathway

Fanconi anemia (FA) patients exhibit bone marrow failure, developmental defects and cancer. The FA pathway maintains chromosomal stability in concert with replication fork maintenance and DNA double strand break (DSB) repair pathways including RAD51-mediated homologous recombination (HR). RAD51 is a recombinase that maintains replication forks and repairs DSBs, but also rearranges chromosomes. Two RecQ helicases, RECQL5 and Bloom syndrome mutated (BLM) suppress HR through nonredundant mechanisms. Here we test the impact deletion of RECQL5 and BLM has on mouse embryonic stem (ES) cells deleted for FANCB, a member of the FA core complex. We show that RECQL5, but not BLM, conferred resistance to mitomycin C (MMC, an interstrand crosslinker) and camptothecin (CPT, a type 1 topoisomerase inhibitor) in FANCB-defective cells. RECQL5 suppressed, while BLM caused, breaks and radials in FANCB-deleted cells exposed to CPT or MMC, respectively. RECQL5 protected the nascent replication strand from MRE11-mediated degradation and restarted stressed replication forks in a manner additive to FANCB. By contrast BLM restarted, but did not protect, replication forks in a manner epistatic to FANCB. RECQL5 also lowered RAD51 levels in FANCB-deleted cells at stressed replication sites implicating a rearrangement avoidance mechanism. Thus, RECQL5 and BLM impact FANCB-defective cells differently in response to replication stress with relevance to chemotherapeutic regimes.     

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity,Leading to the Under Replication of DNA

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.