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Stephen C Kowalczykowski - One of the best experts on this subject based on the ideXlab platform.

  • single molecule visualization of RecQ Helicase reveals dna melting nucleation and assembly are required for processive dna unwinding
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Anthony L Forget, Ronald J Baskin, Stephen C Kowalczykowski
    Abstract:

    DNA Helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ Helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the Helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ Helicase.

  • RecQ Helicase and recj nuclease provide complementary functions to resect dna for homologous recombination
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Katsumi Morimatsu, Stephen C Kowalczykowski
    Abstract:

    Recombinational DNA repair by the RecF pathway of Escherichia coli requires the coordinated activities of RecA, RecFOR, RecQ, RecJ, and single-strand DNA binding (SSB) proteins. These proteins facilitate formation of homologously paired joint molecules between linear double-stranded (dsDNA) and supercoiled DNA. Repair starts with resection of the broken dsDNA by RecQ, a 3′→5′ Helicase, RecJ, a 5′→3′ exonuclease, and SSB protein. The ends of a dsDNA break can be blunt-ended, or they may possess either 5′- or 3′-single-stranded DNA (ssDNA) overhangs of undefined length. Here we show that RecJ nuclease alone can initiate nucleolytic resection of DNA with 5′-ssDNA overhangs, and that RecQ Helicase can initiate resection of DNA with blunt-ends or 3′-ssDNA overhangs by DNA unwinding. We establish that in addition to its well-known ssDNA exonuclease activity, RecJ can display dsDNA exonuclease activity, degrading 100–200 nucleotides of the strand terminating with a 5′-ssDNA overhang. The dsDNA product, with a 3′-ssDNA overhang, is an optimal substrate for RecQ, which unwinds this intermediate to reveal the complementary DNA strand with a 5′-end that is degraded iteratively by RecJ. On the other hand, RecJ cannot resect duplex DNA that is either blunt-ended or terminated with 3′-ssDNA; however, such DNA is unwound by RecQ to create ssDNA for RecJ exonuclease. RecJ requires interaction with SSB for exonucleolytic degradation of ssDNA but not dsDNA. Thus, complementary action by RecJ and RecQ permits initiation of recombinational repair from all dsDNA ends: 5′-overhangs, blunt, or 3′-overhangs. Such Helicase–nuclease coordination is a common mechanism underlying resection in all organisms.

  • translocation of e coli RecQ Helicase on single stranded dna
    Biochemistry, 2012
    Co-Authors: Stephen C Kowalczykowski
    Abstract:

    Helicases couple the hydrolysis of ATP to unidirectional movement on single-stranded DNA (ssDNA). Upon encountering double-stranded DNA (dsDNA), these ATP driven motors force the displacement of the opposite strand (1, 2), allowing essential metabolic processes such as DNA recombination, repair, and replication to proceed. Translocation on ssDNA also serves other biological roles such as displacing bound proteins to regulate pathways such as DNA recombination (3–5). The RecQ Helicase from E. coli is a Super-Family 2 (SF2) Helicase involved in the repair and recombination of DNA (6, 7). This protein is the founding member of the RecQ protein family of Helicases, which is a family of highly conserved motor proteins in bacteria and eukaryotes. RecQ was identified as a gene that conferred resistance to thymine-less growth and was found to function in recombinational DNA repair by the RecF pathway (8). RecQ, in conjunction with RecJ, processes DNA at the site of a stalled replisome to allow daughter-strand gap repair and recombination-mediated restart of DNA replication (9). RecQ, a 3′ to 5′ Helicase (10), and RecJ, a 5′ to 3′ exonuclease (11), can process double-strand breaks to produce 3′-terminated ssDNA (12). This ssDNA serves as the substrate for assembly of a RecA filament, which then finds homology in intact dsDNA and promotes pairing of the ssDNA to the homologous target. RecQ has additional roles in DNA recombination and replication. RecQ can disrupt joint molecules both in vivo (13) and in vitro (12, 14). In addition, RecQ can function with Topoisomerase III (Topo III), a type I topoisomerase, to catenate and decatenate dsDNA (15, 16). This reaction can serve two potential functions: one is to dissolve double Holliday junctions formed during recombination (17–20), and the other is to decatenate converged replication forks (21). In vitro, the RecQ Helicase binds to and unwinds a variety of different DNA substrates (10, 14, 22, 23). DNA with an ssDNA tail, gapped DNA, blunt dsDNA and covalently closed dsDNA are all unwound by RecQ, indicating that the Helicase can function on a variety of intermediates found in DNA metabolism. RecQ is one of the few Helicases that can unwind covalently closed, circular DNA—showing that it does not require a DNA end to enter dsDNA (15, 24). The ssDNA binding protein (SSB) from E. coli interacts with RecQ and stimulates its Helicase activity to the extent that RecQ can unwind plasmid-length DNA (21, 24, 25). Initially, Helicase assays performed with plasmid DNA suggested a nearly stoichiometric mechanism for unwinding dsDNA by RecQ (10, 22). In this mechanism, short patches of dsDNA are unwound by RecQ translocation over relatively short distances (24). However, unwinding of dsDNA is stimulated by SSB. In the presences of SSB, optimal unwinding requires 1 RecQ for every 30 base pairs, indicating that each Helicase is capable of unwinding a region of DNA longer than its DNA binding site size (24, 26). Furthermore, experiments with dsDNA possessing a 3′-ssDNA tail show that RecQ alone can unwind dsDNA up to 25 base-pairs in length (27). The translocation mechanism of RecQ, however, has not been studied in as great detail as the unwinding mechanism. Several assays, as well as models, exist for the study of translocation by Helicases on ssDNA. Steady-state analysis of several Helicases on ssDNA has revealed estimates of processivity, directionality, and sequence effects on translocation (28–30). Kinetic mechanisms of translocation can be elucidated from analysis of the steady-state ATP hydrolysis activity. To understand translocation by RecQ, we studied the steady-state behavior of its ssDNA-dependent ATP hydrolysis activity as a function of DNA length. We find that the activity of RecQ increases with longer lengths of ssDNA. Moreover, when studied on ssDNA that is coated with T4 gene 32 protein (G32P) to limit the size of ssDNA available for activity, ATP hydrolysis was reduced in a manner that is quantitatively consistent with RecQ translocation on the ssDNA gaps that were available. We present a model to describe the mechanism of translocation for RecQ.

  • efficient coupling of atp hydrolysis to translocation by RecQ Helicase
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Stephen C Kowalczykowski
    Abstract:

    Helicases are ubiquitous enzymes that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) during essential processes such as replication, transcription, or repair. The Escherichia coli RecQ protein is a 3′ to 5′ Helicase, which functions in the processes of homologous recombination and replication fork restart. Here, we analyzed the relationship between ATP hydrolysis by RecQ and its translocation on ssDNA. We monitored a single round of RecQ translocation on ssDNA by measuring the rates of inorganic phosphate release during translocation, and the dissociation of RecQ from ssDNA. We find that RecQ translocates with a rate of 16( ± 4) nucleotides/s and moves on average only 36( ± 2) nucleotides before dissociating. Fitting to an n-step kinetic model suggests that the Helicase displays a nonuniform translocation mechanism in which it moves approximately five nucleotides rapidly before undergoing a rate-limiting kinetic slow step. Unexpectedly, RecQ requires a length of 34( ± 3) nucleotides to bind and translocate on ssDNA. This large site size suggests that several monomers are required to bind DNA prior to translocation. Energetically, the RecQ Helicase couples the hydrolysis of one ATP molecule to the translocation of more than one nucleotide (1.6 ± 0.3). Thus, our data show that RecQ translocates on ssDNA by efficiently coupling the hydrolysis of one ATP molecule into structural alterations that result in movement of approximately two nucleotides, presumably by an inchworm mechanism. These attributes are consistent with the function of RecQ in recombination and replication.

  • RecQ Helicase stimulates both dna catenation and changes in dna topology by topoisomerase iii
    Journal of Biological Chemistry, 2003
    Co-Authors: Frank G Harmon, Joel P Brockman, Stephen C Kowalczykowski
    Abstract:

    Together, RecQ Helicase and topoisomerase III (Topo III) of Escherichia coli comprise a potent DNA strand passage activity that can catenate covalently closed DNA (Harmon, F. G., DiGate, R. J., and Kowalczykowski, S. C. (1999) Mol. Cell 3, 611-620). Here we directly assessed the structure of the catenated DNA species formed by RecQ Helicase and Topo III using atomic force microscopy. The images show complex catenated DNA species involving crossovers between multiple double-stranded DNA molecules that are consistent with full catenanes. E. coli single-stranded DNA-binding protein significantly stimulated both the topoisomerase activity of Topo III alone and the DNA strand passage activity of RecQ Helicase and Topo III. Titration data suggest that an intermediate of the RecQ Helicase unwinding process, perhaps a RecQ Helicase-DNA fork, is the target for Topo III action. Catenated DNA is the predominant product under conditions of molecular crowding; however, we also discovered that RecQ Helicase and single-stranded DNA-binding protein greatly stimulated the intramolecular strand passage ("supercoiling") activity of Topo III, as revealed by changes in the linking number of uncatenated DNA. Together our results demonstrate that RecQ Helicase and Topo III function together to comprise a potent and concerted single-strand DNA passage activity that can mediate both catenation-decatenation processes and changes in DNA topology.

Ian D. Hickson - One of the best experts on this subject based on the ideXlab platform.

  • yeast as a model system to study RecQ Helicase function
    DNA Repair, 2010
    Co-Authors: Thomas M Ashton, Ian D. Hickson
    Abstract:

    Abstract Mutations in the highly conserved RecQ Helicase, BLM , cause the rare cancer predisposition disorder, Bloom's syndrome. The orthologues of BLM in Saccharomyces cerevisiae and Schizosaccharomyces pombe are SGS1 and rqh1 + , respectively. Studies in these yeast species have revealed a plethora of roles for the Sgs1 and Rqh1 proteins in repair of double strand breaks, restart of stalled replication forks, processing of aberrant intermediates that arise during meiotic recombination, and maintenance of telomeres. In this review, we focus on the known roles of Sgs1 and Rqh1 and how studies in yeast species have improved our knowledge of how BLM suppresses neoplastic transformation.

  • RecQ Helicases: multifunctional genome caretakers
    Nature Reviews Cancer, 2009
    Co-Authors: Ian D. Hickson
    Abstract:

    In recent years, there has been a tremendous advance in our understanding of the cellular functions of individual RecQ Helicases. This Review discusses how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors. RecQ Helicases are a highly conserved family and are considered to be genome 'caretakers' that maintain chromosome stability and suppress neoplastic transformation. The crystal structure of RecQ Helicases reveals several conserved functional domains. Some of these domains define a particular member of the RecQ Helicase family and may be important for functionally distinguishing between the different RecQ Helicases expressed in a particular organism. Defects in three of the five human RecQ Helicase members give rise to at least five defined disorders associated with cancer predisposition, premature ageing and developmental abnormalities. Mutations in BLM and WRN lead to Bloom's and Werner's syndromes, respectively, and RecQ4 is associated with three distinct disorders: Rothmund–Thomson, RAPADILINO and Baller–Gerold syndromes. These RecQ4 disorders display the common feature of an abnormality in bone development. Mouse models of human RecQ Helicase disorders have been generated that partially recapitulate the phenotypes seen in human patients. RecQ Helicases possess several biochemical activities and have several important roles in DNA replication and recombination. Some of these activities require or are modulated by physical interactions with other nuclear proteins. BLM is proposed to have roles in mitosis that resolve late-replication intermediates in conjunction with topoisomerase IIIα. Together, these proteins probably function to decatenate entangled DNA that arises during DNA replication. Around 1% of the open reading frames in the human genome encode predicted DNA and RNA Helicases. One highly conserved group of DNA Helicases is the RecQ family. Genetic defects in three of the five human RecQ Helicases, BLM, WRN and RecQ4, give rise to defined syndromes associated with cancer predisposition, some features of premature ageing and chromosomal instability. In recent years, there has been a tremendous advance in our understanding of the cellular functions of individual RecQ Helicases. In this Review, we discuss how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.

  • the RecQ Helicase topoisomerase iii rmi1 complex a dna structure specific dissolvasome
    Trends in Biochemical Sciences, 2007
    Co-Authors: Hocine W Mankouri, Ian D. Hickson
    Abstract:

    RecQ Helicases, together with topoisomerase III and Rmi1 family proteins, form an evolutionarily conserved complex that is essential for the maintenance of genome integrity. This complex, which we term RTR, is capable of, or has been implicated in, the processing of a diverse array of DNA structures, and we propose here that it functions in a coordinated fashion as a DNA structure-specific ‘dissolvasome'. Little is known about how the RTR complex might be regulated or targeted to various DNA structures in vivo . Recent findings indicate that the components of the RTR complex might activate the cell cycle checkpoint machinery as well as be a target of checkpoint kinases, suggesting that these events are crucial to ensure faithful DNA replication and chromosome segregation.

  • The RecQ Helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific 'dissolvasome'?
    Trends in Biochemical Sciences, 2007
    Co-Authors: Hocine W Mankouri, Ian D. Hickson
    Abstract:

    RecQ Helicases, together with topoisomerase III and Rmi1 family proteins, form an evolutionarily conserved complex that is essential for the maintenance of genome integrity. This complex, which we term RTR, is capable of, or has been implicated in, the processing of a diverse array of DNA structures, and we propose here that it functions in a coordinated fashion as a DNA structure-specific 'dissolvasome'. Little is known about how the RTR complex might be regulated or targeted to various DNA structures in vivo. Recent findings indicate that the components of the RTR complex might activate the cell cycle checkpoint machinery as well as be a target of checkpoint kinases, suggesting that these events are crucial to ensure faithful DNA replication and chromosome segregation.

  • Understanding the roles of RecQ Helicases in the maintenance of genome integrity and suppression of tumorigenesis
    Biochemical Society Transactions, 2004
    Co-Authors: Hocine W Mankouri, Ian D. Hickson
    Abstract:

    RecQ Helicases are evolutionarily conserved enzymes required for the maintenance of genome stability. Mutations in three of the five known human RecQ Helicase genes cause distinct clinical disorders that are characterized by genome instability and cancer predisposition. Recent studies have begun to reveal the cellular roles of RecQ Helicases and how these enzymes may prevent tumorigenesis at the molecular level.

Keir C Neuman - One of the best experts on this subject based on the ideXlab platform.

  • RecQ Helicase triggers a binding mode change in the ssb dna complex to efficiently initiate dna unwinding
    Nucleic Acids Research, 2017
    Co-Authors: Maria Mills, Yeonee Seol, Mihaly Kovacs, Mate Gyimesi, Gabor M Harami, Mate Martina, Zoltan Kovacs, Keir C Neuman
    Abstract:

    : The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ Helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.

  • single molecule measurements of dna decatenation by the topoisomerase iii RecQ Helicase complex
    Biophysical Journal, 2016
    Co-Authors: Maria K Mills, Keir C Neuman
    Abstract:

    Topoisomerase III (Topo III) is an ATP-independent enzyme that cleaves a single strand of duplex DNA to relieve torsional strain. Topo III is able to unlink DNA catenanes if one strand contains a single stranded region, but not if both strands are intact. Topo III, however, can decatenate intact DNA when RecQ Helicase is present. In vivo this complex is involved in resolution of late replication intermediate linkages and double Holliday junctions. More generally, the unique functions of the Topo III-RecQ complex have been shown to be important for preserving genome stability.We directly measured the effect of RecQ on Topo III's decatenase activity using a magnetic tweezer assay in which two strands of DNA attached to a single bead are interwound to produce a DNA braid. These DNA braids topologically mimic catenated DNA and thus provide an ideal substrate for measuring unlinking at the single molecule level. We tested the effect of RecQ on the rate of Topo III decatenation and unwinding activity. In addition we measured decatenation of intact DNA by the RecQ-Topo III complex. We compared these results to the effect of RecQ on two other topoisomerases that decatenate DNA but are not known to interact with RecQ: topoisomerase I, which acts on DNA with a single stranded region, and topoisomerase IV, which acts on intact DNA. We also probed the effect of crossover geometry on the decatenation activity of the Topo III-RecQ complex. Our results provide insight into the functional interactions between Topo III and RecQ.

  • the hrdc domain of e coli RecQ Helicase controls single stranded dna translocation and double stranded dna unwinding rates without affecting mechanoenzymatic coupling
    Scientific Reports, 2015
    Co-Authors: Gabor M Harami, Keir C Neuman, Mate Martina, Nikolett T Nagy, Mihaly Kovacs
    Abstract:

    DNA-restructuring activities of RecQ-family Helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the Helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom’s syndrome (BLM) Helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ Helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

  • functional implications of the RecQ Helicase topoisomerase iii ssb complex insights from single molecule measurements
    Biophysical Journal, 2015
    Co-Authors: Maria K Mills, Yeonee Seol, Keir C Neuman
    Abstract:

    RecQ Helicases are a highly conserved class of ATP-dependent DNA Helicases that perform multifunctional roles in genome maintenance. In E. coli, RecQ is known to physically and functionally interact with Topoisomerase III, a type IA topoisomerase. The coupling of Helicase activity and topoisomerase activity that results from this interaction is responsible for resolving complex DNA structures such as double Holliday junctions. Similar interactions have been demonstrated in homologous proteins in other organisms, including the human RecQ Helicase BLM and TopoIIIα, and the yeast Helicase SgsI and TopoIII. There is also evidence that single-stranded DNA binding protein (SSB) interacts with RecQ and is a necessary component of this complex in vivo. We sought to explore the mechanism by which RecQ stimulates TopoIII activity, and by which SSB stimulates RecQ activity, as well as the individual contributions of these proteins to topological changes in DNA using single molecule experiments. To investigate the roles of RecQ, TopoIII, and SSB, individually and in a ternary complex, we measured their effect on DNA hairpin unwinding and refolding using magnetic tweezers. We also conducted experiments on over- and under-wound double-stranded DNA to probe the roles of RecQ and SSB in activation of TopoIII relaxation of supercoiled DNA. This technique allows us to probe changes in DNA topology as a consequence of enzyme activity in real time. Our findings demonstrate a complex set of interactions and provide a framework for understanding the mechanism of the resolvase activity of the RecQ-TopoIII- SSB complex.

  • domain architecture of RecQ Helicase defines mechanochemical linkage via multipartite interactions with dna substrate during unwinding activity
    Biophysical Journal, 2014
    Co-Authors: Gabor M Harami, Junghoon In, Yeonee Seol, Kata Sarlos, Keir C Neuman, Mate Martina, Mihaly Kovacs
    Abstract:

    RecQ Helicases are ubiquitous enzymes that safeguard the genome by playing multiple roles in DNA repair, replication and recombination. They are unique in their capability to process a wide range of non-canonical structures associated with DNA metabolic intermediates. DNA-binding domains linked to the RecQ Helicase core, including the winged-helix (WHD) and Helicase-and-RnaseD-C-terminal (HRDC) domains, are thought to confer substrate specificity and modulate enzymatic activities. We combined ensemble biophysical and single-molecule magnetic tweezers assays to determine the mechanochemical linkage between ATP hydrolytic and DNA-restructuring activities of E. coli RecQ constructs of varying domain architecture. We compared the activities of wild-type, HRDC point mutant and deletion mutant, and WHD-HRDC deletion mutant constructs using several defined DNA structures and experimental geometries. We show that the WHD enhances unwinding processivity by stabilizing enzyme-DNA interactions, whereas the HRDC domain increases the overall DNA affinity but hinders the unwinding and ATPase activities of the Helicase through interactions with single-stranded (ss) DNA regions. Intriguingly, HRDC-ssDNA interactions stabilize the pausing of the Helicase during unwinding of a DNA hairpin in which both nascent ssDNA strands are mechanically strained, but not that of gapped duplex DNA in which one of the liberated ssDNA strands is mechanically unconstrained. Our study reveals that both the DNA substrate geometry and the contribution of auxiliary DNA-binding domains greatly influence the mechanochemical behavior of the Helicase and the outcome of DNA-processing reactions, bearing consequences on the diverse in vivo actions of RecQ enzymes.

Raymond J Monnat - One of the best experts on this subject based on the ideXlab platform.

  • human RecQ Helicase pathogenic variants population variation and missing diseases
    Human Mutation, 2017
    Co-Authors: Wenqing Fu, Alessio Ligabue, Kai J Rogers, Joshua M Akey, Raymond J Monnat
    Abstract:

    Heritable loss of function mutations in the human RecQ Helicase genes BLM, WRN and RecQL4 cause Bloom, Werner and Rothmund-Thomson syndromes, cancer predispositions with additional developmental or progeroid features. In order to better understand RecQ pathogenic and population variation, we systematically analyzed genetic variation in all five human RecQ Helicase genes. A total of 3,741 unique basepair-level variants were identified, across 17,605 potential mutation sites. Direct counting of BLM, RecQL4 and WRN pathogenic variants was used to determine aggregate and disease-specific carrier frequencies. The use of biochemical and model organism data, together with computational prediction, identified over 300 potentially pathogenic population variants in RecQL and RecQL5, the two RecQ Helicases that are not yet linked to a heritable deficiency syndrome. Despite the presence of these predicted pathogenic variants in the human population, we identified no individuals homozygous for any biochemically-verified or predicted pathogenic RecQL or RecQL5 variant. Nor did we find any individual heterozygous for known pathogenic variants in two or more of the disease-associated RecQ Helicase genes BLM, RecQL4 or WRN. Several postulated RecQ Helicase deficiency syndromes–RecQL or RecQL5 loss of function, or compound haplo-insufficiency for the disease-associated RecQ Helicases–may remain missing, as they likely incompatible with life. This article is protected by copyright. All rights reserved

  • the werner syndrome RecQ Helicase targets g4 dna in human cells to modulate transcription
    Human Molecular Genetics, 2016
    Co-Authors: Weiliang Tang, Junko Oshima, Nancy Maizels, Ana I Robles, Richard P Beyer, Lucas T Gray, G Nguyen, Curtis C Harris, Raymond J Monnat
    Abstract:

    The Werner syndrome (WS) is a prototypic adult Mendelian progeroid syndrome in which signs of premature aging are associated with genomic instability and an elevated risk of cancer. The WRN RecQ Helicase protein binds and unwinds G-quadruplex (G4) DNA substrates in vitro, and we identified significant enrichment in G4 sequence motifs at the transcription start site and 5' ends of first introns (false discovery rate < 0.001) of genes down-regulated in WS patient fibroblasts. This finding provides strong evidence that WRN binds G4 DNA structures at many chromosomal sites to modulate gene expression. WRN appears to bind a distinct subpopulation of G4 motifs in human cells, when compared with the related Bloom syndrome RecQ Helicase protein. Functional annotation of the genes and miRNAs altered in WS provided new insight into WS disease pathogenesis. WS patient fibroblasts displayed altered expression of multiple, mechanistically distinct, senescence-associated gene expression programs, with altered expression of disease-associated miRNAs, and dysregulation of canonical pathways that regulate cell signaling, genome stability and tumorigenesis. WS fibroblasts also displayed a highly statistically significant and distinct gene expression signature, with coordinate overexpression of nearly all of the cytoplasmic tRNA synthetases and associated ARS-interacting multifunctional protein genes. The 'non-canonical' functions of many of these upregulated tRNA charging proteins may together promote WS disease pathogenesis. Our results identify the human WRN RecQ protein as a G4 Helicase that modulates gene expression in G4-dependent fashion at many chromosomal sites and provide several new and unexpected mechanistic insights into WS disease pathogenesis.

  • altered RecQ Helicase expression in sporadic primary colorectal cancers
    Translational Oncology, 2013
    Co-Authors: Piri Welcsh, Raymond J Monnat, Kelly T Carter, Slavomir Dzieciatkowski, Suzanne M Dintzis, Jane L Meza, Nora Sarvetnick, Lawrence A Loeb, William M Grady
    Abstract:

    Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RecQ Helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RecQ Helicase (WRN, BLM, RecQL, RecQL4, and RecQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RecQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RecQL and RecQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RecQ Helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RecQ Helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RecQ Helicase expression. Immunohistochemistry localized the RecQ Helicases to the nucleus. RecQ Helicase expression is altered in CRC, suggesting that RecQ Helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.

  • human RecQ Helicases roles in dna metabolism mutagenesis and cancer biology
    Seminars in Cancer Biology, 2010
    Co-Authors: Raymond J Monnat
    Abstract:

    Helicases use the energy of ATP hydrolysis to separate double-stranded nucleic acids to facilitate essential processes such as replication, recombination, transcription and repair. This article focuses on the human RecQ Helicase gene and protein family. Loss of function of three different members has been shown to cause Bloom syndrome (BS), Werner syndrome (WS) and Rothmund–Thomson syndrome (RTS). This article outlines clinical and cellular features of these cancer predisposition syndromes, and discusses their pathogenesis in light of our understanding of RecQ Helicase biochemical activities and in vivo functions. I also discuss the emerging role for RecQ Helicases as predictors of disease risk and the response to therapy.

  • functional role of the werner syndrome RecQ Helicase in human fibroblasts
    Aging Cell, 2007
    Co-Authors: Kiranjit K Dhillon, Julia M Sidorova, Peter S. Rabinovitch, Yannick Saintigny, Martin Poot, Katherine A Gollahon, Raymond J Monnat
    Abstract:

    Summary Werner syndrome is an autosomal recessive human genetic instability and cancer predisposition syndrome that also has features of premature aging. We focused on two questions related to Werner syndrome protein (WRN) function in human fibroblasts: Do WRN-deficient fibroblasts have a consistent cellular phenotype? What role does WRN play in the recovery from replication arrest? We identified consistent cell proliferation and DNA damage sensitivity defects in both primary and SV40-transformed fibroblasts from different Werner syndrome patients, and showed that these defects could be revealed by acute depletion of WRN protein. Mechanistic analysis of the role of WRN in recovery from replication arrest indicated that WRN acts to repair damage resulting from replication arrest, rather than to prevent the disruption or breakage of stalled replication forks. These results identify readily quantified cell pheno- types that result from WRN loss in human fibroblasts; delineate the impact of cell transformation on the expres- sion of these phenotypes; and define a mechanistic role for WRN in the recovery from replication arrest.

Mihaly Kovacs - One of the best experts on this subject based on the ideXlab platform.

  • RecQ Helicase triggers a binding mode change in the ssb dna complex to efficiently initiate dna unwinding
    Nucleic Acids Research, 2017
    Co-Authors: Maria Mills, Yeonee Seol, Mihaly Kovacs, Mate Gyimesi, Gabor M Harami, Mate Martina, Zoltan Kovacs, Keir C Neuman
    Abstract:

    : The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ Helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.

  • the hrdc domain of e coli RecQ Helicase controls single stranded dna translocation and double stranded dna unwinding rates without affecting mechanoenzymatic coupling
    Scientific Reports, 2015
    Co-Authors: Gabor M Harami, Keir C Neuman, Mate Martina, Nikolett T Nagy, Mihaly Kovacs
    Abstract:

    DNA-restructuring activities of RecQ-family Helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the Helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom’s syndrome (BLM) Helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ Helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

  • mechanism of RecQ Helicase mechanoenzymatic coupling reveals that the dna interactions of the adp bound enzyme control translocation run terminations
    Nucleic Acids Research, 2015
    Co-Authors: Kata Sarlos, Mate Gyimesi, Zoltan Kele, Mihaly Kovacs
    Abstract:

    The processing of various DNA structures by RecQ Helicases is crucial for genome maintenance in both bacteria and eukaryotes. RecQ Helicases perform active destabilization of DNA duplexes, based on tight coupling of their ATPase activity to moderately processive translocation along DNA strands. Here, we determined the ATPase kinetic mechanism of E. coli RecQ Helicase to reveal how mechanoenzymatic coupling is achieved. We found that the interaction of RecQ with DNA results in a drastic acceleration of the rate-limiting ATP cleavage step, which occurs productively due to subsequent rapid phosphate release. ADP release is not rate-limiting and ADP-bound RecQ molecules make up a small fraction during single-stranded DNA translocation. However, the relatively rapid release of the ADP-bound enzyme from DNA causes the majority of translocation run terminations (i.e. detachment from the DNA track). Thus, the DNA interactions of ADP-bound RecQ Helicase, probably dependent on DNA structure, will mainly determine translocation processivity and may control the outcome of DNA processing. Comparison with human Bloom's syndrome (BLM) Helicase reveals that similar macroscopic parameters are achieved by markedly different underlying mechanisms of RecQ homologs, suggesting diversity in enzymatic tuning.

  • a nucleotide dependent and hrdc domain dependent structural transition in dna bound RecQ Helicase
    Journal of Biological Chemistry, 2014
    Co-Authors: Zsuzsa S Kocsis, Kata Sarlos, Gabor M Harami, Mate Martina, Mihaly Kovacs
    Abstract:

    The allosteric communication between the ATP- and DNA-binding sites of RecQ Helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ Helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound Helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the Helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ Helicases.

  • domain architecture of RecQ Helicase defines mechanochemical linkage via multipartite interactions with dna substrate during unwinding activity
    Biophysical Journal, 2014
    Co-Authors: Gabor M Harami, Junghoon In, Yeonee Seol, Kata Sarlos, Keir C Neuman, Mate Martina, Mihaly Kovacs
    Abstract:

    RecQ Helicases are ubiquitous enzymes that safeguard the genome by playing multiple roles in DNA repair, replication and recombination. They are unique in their capability to process a wide range of non-canonical structures associated with DNA metabolic intermediates. DNA-binding domains linked to the RecQ Helicase core, including the winged-helix (WHD) and Helicase-and-RnaseD-C-terminal (HRDC) domains, are thought to confer substrate specificity and modulate enzymatic activities. We combined ensemble biophysical and single-molecule magnetic tweezers assays to determine the mechanochemical linkage between ATP hydrolytic and DNA-restructuring activities of E. coli RecQ constructs of varying domain architecture. We compared the activities of wild-type, HRDC point mutant and deletion mutant, and WHD-HRDC deletion mutant constructs using several defined DNA structures and experimental geometries. We show that the WHD enhances unwinding processivity by stabilizing enzyme-DNA interactions, whereas the HRDC domain increases the overall DNA affinity but hinders the unwinding and ATPase activities of the Helicase through interactions with single-stranded (ss) DNA regions. Intriguingly, HRDC-ssDNA interactions stabilize the pausing of the Helicase during unwinding of a DNA hairpin in which both nascent ssDNA strands are mechanically strained, but not that of gapped duplex DNA in which one of the liberated ssDNA strands is mechanically unconstrained. Our study reveals that both the DNA substrate geometry and the contribution of auxiliary DNA-binding domains greatly influence the mechanochemical behavior of the Helicase and the outcome of DNA-processing reactions, bearing consequences on the diverse in vivo actions of RecQ enzymes.