Human RecQ helicases in DNA repair, recombination, and replication.

RecQ helicases are an important family of genome surveillance proteins conserved from bacteria to humans. Each of the five human RecQ helicases plays critical roles in genome maintenance and stability, and the RecQ protein family members are often referred to as guardians of the genome. The importance of these proteins in cellular homeostasis is underscored by the fact that defects in BLM, WRN, and RECQL4 are linked to distinct heritable human disease syndromes. Each human RecQ helicase has a unique set of protein-interacting partners, and these interactions dictate its specialized functions in genome maintenance, including DNA repair, recombination, replication, and transcription. Human RecQ helicases also interact with each other, and these interactions have significant impact on enzyme function. Future research goals in this field include a better understanding of the division of labor among the human RecQ helicases and learning how human RecQ helicases collaborate and cooperate to enhance genome stability.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

TOPping off meiosis.

Double-strand breaks (DSBs) threaten chromosome integrity. The most accurate repair of DSBs is by homologous recombination (HR), catalyzed by recombination proteins such as Rad51. Three papers in this issue of Molecular Cell (Fasching et al., 2015; Kaur et al., 2015; Tang et al., 2015) now reveal the role of three of these proteins in budding yeast: Sgs1 (BLM homolog), Top3 (TOPIIIα homolog), and Rmi1. They demonstrate several steps where all three proteins act together, and find additional functions of the Top3-Rmi1 subcomplex that are critical for the completion of meiosis.

Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.

The displacement loop (D loop) is a DNA strand invasion product formed during homologous recombination. Disruption of nascent D loops prevents recombination, and during synthesis-dependent strand annealing (SDSA), disruption of D loops extended by DNA polymerase ensures a non-crossover outcome. The proteins implicated in D loop disruption are DNA motor proteins/helicases that act by moving DNA junctions. Here we report that D loops can also be disrupted by DNA topoisomerase 3 (Top3), and this disruption depends on Top3's catalytic activity. Yeast Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54; protein-free D loops or D loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also, the human Topoisomerase IIIa-RMI1-RMI2 complex is capable of dissolving D loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is partially a result of unprocessed D loops.

A distinct role for recombination repair factors in an early cellular response to transcription-replication conflicts.

Transcription-replication (T-R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T-R conflicts, our understanding of the genetic and temporal nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T-R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-independent function of FANCD2. A hallmark of the TRe response is the rapid co-localization of these three DNA repair factors at sites of T-R collisions. We find that the TRe response relies on basal activity of the ATR kinase, yet it does not lead to hyperactivation of this key checkpoint protein. Furthermore, specific abrogation of the TRe response leads to DNA damage in mitosis, and promotes chromosome instability and cell death. Collectively our findings identify a new role for these well-established tumor suppressor proteins at an early stage of the cellular response to conflicts between DNA transcription and replication.

The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair.

DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)- and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.

Alternative lengthening of telomeres (ALT) is a telomere lengthening pathway that predominates in aggressive tumors of mesenchymal origin; however, the underlying mechanism of telomere synthesis is not fully understood. Here, we show that the BLM-TOP3A-RMI (BTR) dissolvase complex is required for ALT-mediated telomere synthesis. We propose that recombination intermediates formed during strand invasion are processed by the BTR complex, initiating rapid and extensive POLD3-dependent telomere synthesis followed by dissolution, with no overall exchange of telomeric DNA. This process is counteracted by the SLX4-SLX1-ERCC4 complex, which promotes resolution of the recombination intermediate, resulting in telomere exchange in the absence of telomere extension. Our data are consistent with ALT being a conservative DNA replication process, analogous to break-induced replication, which is dependent on BTR and counteracted by SLX4 complex-mediated resolution events.

Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB.

Most RecQ DNA helicases share a conserved domain arrangement that mediates their activities in genomic stability. This arrangement comprises a helicase motor domain, a RecQ C-terminal (RecQ-C) region including a winged-helix (WH) domain, and a 'Helicase and RNase D C-terminal' (HRDC) domain. Single-molecule real-time translocation and DNA unwinding by full-length Escherichia coli RecQ and variants lacking either the HRDC or both the WH and HRDC domains was analyzed. RecQ operated under two interconvertible kinetic modes, 'slow' and 'normal', as it unwound duplex DNA and translocated on single-stranded (ss) DNA. Consistent with a crystal structure of bacterial RecQ bound to ssDNA by base stacking, abasic sites blocked RecQ unwinding. Removal of the HRDC domain eliminates the slow mode while preserving the normal mode of activity. Unexpectedly, a RecQ variant lacking both the WH and HRDC domains retains weak helicase activity. The inclusion of E. coli ssDNA-binding protein (SSB) induces a third 'fast' unwinding mode four times faster than the normal RecQ mode and enhances the overall helicase activity (affinity, rate, and processivity). SSB stimulation was, furthermore, observed in the RecQ deletion variants, including the variant missing the WH domain. Our results support a model in which RecQ and SSB have multiple interacting modes.

BLM can regulate cataract progression by influencing cell vitality and apoptosis.

Age-related cataract (ARC) is the most common cause of severe visual impairment and blindness. The precise mechanisms of ARC are not completely understood, but it is well accepted that oxidative damage plays an important role in the disease pathogenesis. BLM, the key enzyme of the double-strand break repair (DSBR) pathway, is part of a family of DNA unwinding enzymes and has a crucial role in multiple steps of the DNA recombination, replication and repair processes. We have recently shown that BLM-rs1063147 is initially associated with nuclear ARC in a cross-section study. Therefore, we wanted to study the effects of BLM on ARC progression. In ARC patients, BLM transcription in lens capsules was decreased, so did the BLM protein, and after UVB irradiation, BLM mRNA and protein levels were increased in SRA01/04 cells. Upon silencing BLM in SRA01/04 cells and rat lens, cell vitality and apoptosis were altered, and the rat lens opacification was considerable. In conclusion, BLM can regulate cataract progression by influencing cell vitality and apoptosis.

Degradation of Mrc1 promotes recombination-mediated restart of stalled replication forks.

The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. Replication stress or DNA damage triggers fork stalling and checkpoint signaling to activate repair pathways. Recovery from checkpoint activation is critical for cell survival following DNA damage. Recovery from the S-phase checkpoint includes inactivation of checkpoint signaling and restart of stalled replication forks. Previous studies demonstrated that degradation of Mrc1, the Saccharomyces cerevisiae ortholog of human Claspin, is facilitated by the SCFDia2 ubiquitin ligase and is important for cell cycle re-entry after DNA damage-induced S-phase checkpoint activation. Here, we show that degradation of Mrc1 facilitated by the SCFDia2 complex is critical to restart stalled replication forks during checkpoint recovery. Using DNA fiber analysis, we showed that Dia2 functions with the Sgs1 and Mph1 helicases (orthologs of human BLM and FANCM, respectively) in the recombination-mediated fork restart pathway. In addition, Dia2 physically interacts with Sgs1 upon checkpoint activation. Importantly, failure to target Mrc1 for degradation during recovery inhibits Sgs1 chromatin association, but this can be alleviated by induced proteolysis of Mrc1 after checkpoint activation. Together, these studies provide new mechanistic insights into how cells recover from activation of the S-phase checkpoint.

BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.

Repair of dsDNA breaks requires processing to produce 3'-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5' → 3' resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells.

Overexpression of BLM promotes DNA damage and increased sensitivity to platinum salts in triple-negative breast and serous ovarian cancers.

Background: Platinum-based therapy is an effective treatment for a subset of triple-negative breast cancer and ovarian cancer patients. In order to increase response rate and decrease unnecessary use, robust biomarkers that predict response to therapy are needed. Patients and methods: We performed an integrated genomic approach combining differential analysis of gene expression and DNA copy number in sensitive compared with resistant triple-negative breast cancers in two independent neoadjuvant cisplatin-treated cohorts. Functional relevance of significant hits was investigated in vitro by overexpression, knockdown and targeted inhibitor treatment. Results: We identified two genes, the Bloom helicase (BLM) and Fanconi anemia complementation group I (FANCI), that have both increased DNA copy number and gene expression in the platinum-sensitive cases. Increased level of expression of these two genes was also associated with platinum but not with taxane response in ovarian cancer. As a functional validation, we found that overexpression of BLM promotes DNA damage and induces sensitivity to cisplatin but has no effect on paclitaxel sensitivity. Conclusions: A biomarker based on the expression levels of the BLM and FANCI genes is a potential predictor of platinum sensitivity in triple-negative breast cancer and ovarian cancer.

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.

Knockout mouse production assisted by Blm knockdown.

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3-4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.

Human RECQL1 participates in telomere maintenance.

A variety of human tumors employ alternative and recombination-mediated lengthening for telomere maintenance (ALT). Human RecQ helicases, such as BLM and WRN, can efficiently unwind alternate/secondary structures during telomere replication and/or recombination. Here, we report a novel role for RECQL1, the most abundant human RecQ helicase but functionally least studied, in telomere maintenance. RECQL1 associates with telomeres in ALT cells and actively resolves telomeric D-loops and Holliday junction substrates. RECQL1 physically and functionally interacts with telomere repeat-binding factor 2 that in turn regulates its helicase activity on telomeric substrates. The telomeric single-stranded binding protein, protection of telomeres 1 efficiently stimulates RECQL1 on telomeric substrates containing thymine glycol, a replicative blocking lesion. Loss of RECQL1 results in dysfunctional telomeres, telomere loss and telomere shortening, elevation of telomere sister-chromatid exchanges and increased aphidicolin-induced telomere fragility, indicating a role for RECQL1 in telomere maintenance. Further, our results indicate that RECQL1 may participate in the same pathway as WRN, probably in telomere replication.

Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress.

Werner syndrome (WS) is a human chromosomal instability disorder associated with cancer predisposition and caused by mutations in the WRN gene. WRN helicase activity is crucial in limiting breakage at common fragile sites (CFS), which are the preferential targets of genome instability in precancerous lesions. However, the precise function of WRN in response to mild replication stress, like that commonly used to induce breaks at CFS, is still missing. Here, we establish that WRN plays a role in mediating CHK1 activation under moderate replication stress. We provide evidence that phosphorylation of CHK1 relies on the ATR-mediated phosphorylation of WRN, but not on WRN helicase activity. Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress. Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels. Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility.

Tumor suppressor RecQL5 controls recombination induced by DNA crosslinking agents.

RecQ family DNA helicases function in the maintenance of genome stability. Mice deficient in RecQL5, one of five RecQ helicases, show a cancer predisposition phenotype, suggesting that RecQL5 plays a tumor suppressor role. RecQL5 interacts with Rad51, a key factor in homologous recombination (HR), and displaces Rad51 from Rad51-single stranded DNA (ssDNA) filaments in vitro. However, the precise roles of RecQL5 in the cell remain elusive. Here, we present evidence suggesting that RecQL5 is involved in DNA interstrand crosslink (ICL) repair. Chicken DT40 RECQL5 gene knockout (KO) cells showed sensitivity to ICL-inducing agents such as cisplatin (CDDP) and mitomycin C (MMC) and a higher number of chromosome aberrations in the presence of MMC than wild-type cells. The phenotypes of RECQL5 KO cells resembled those of Fanconi anemia gene KO cells. Genetic analysis using corresponding gene knockout cells showed that RecQL5 is involved in the FANCD1 (BRCA2)-dependent ICL repair pathway in which Rad51-ssDNA filament formation is promoted by BRCA2. The disappearance but not appearance of Rad51-foci was delayed in RECQL5 KO cells after MMC treatment. Deletion of Rad54, which processes the Rad51-ssDNA filament in HR, in RECQL5 KO cells increased sensitivity to CDDP and further delayed the disappearance of Rad51-foci, suggesting that RecQL5 and Rad54 have different effects on the Rad51-ssDNA filament. Furthermore, the frequency and variation of CDDP-induced gene conversion at the immunoglobulin locus were increased in RECQL5 KO cells. These results suggest that RecQL5 plays a role in regulating the incidence and quality of ICL-induced recombination.

PICH and BLM limit histone association with anaphase centromeric DNA threads and promote their resolution.

Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.

RECQL5 plays co-operative and complementary roles with WRN syndrome helicase.

Humans have five RecQ helicases, whereas simpler organisms have only one. Little is known about whether and how these RecQ helicases co-operate and/or complement each other in response to cellular stress. Here we show that RECQL5 associates longer at laser-induced DNA double-strand breaks in the absence of Werner syndrome (WRN) protein, and that it interacts physically and functionally with WRN both in vivo and in vitro. RECQL5 co-operates with WRN on synthetic stalled replication fork-like structures and stimulates its helicase activity on DNA fork duplexes. Both RECQL5 and WRN re-localize from the nucleolus into the nucleus after replicative stress and significantly associate with each other during S-phase. Further, we show that RECQL5 is essential for cell survival in the absence of WRN. Loss of both RECQL5 and WRN severely compromises DNA replication, accumulates genomic instability and ultimately leads to cell death. Collectively, our results indicate that RECQL5 plays both co-operative and complementary roles with WRN. This is an early demonstration of a significant functional interplay and a novel synthetic lethal interaction among the human RecQ helicases.

Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex.

Mus81 resolvase and Sgs1 helicase have well-established roles in mitotic DNA repair. Moreover, Mus81 is part of a minor crossover (CO) pathway in the meiosis of budding yeast, plants and vertebrates. The major pathway depends on meiosis-specific synaptonemal complex (SC) formation, ZMM proteins and the MutLγ complex for CO-directed resolution of joint molecule (JM)-recombination intermediates. Sgs1 has also been implicated in this pathway, although it may mainly promote the non-CO outcome of meiotic repair. We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions. Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena. Sgs1 may exert functions similar to those in other eukaryotes. However, we propose an additional role in supporting homologous CO formation by promoting homologous over intersister interactions. Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast. We propose that in these two organisms, which independently lost the SC during evolution, the basal set of mitotic repair proteins is sufficient for executing meiotic recombination.