scholarly journals RecBCD possesses strong coupling between DNA and nucleotide binding that may propel a stepping mechanism during translocation

2017 ◽  
Author(s):  
Vera Gaydar ◽  
Rani Zananiri ◽  
Or Dvir ◽  
Ariel Kaplan ◽  
Arnon Henn
Keyword(s):  
Nucleotide Binding ◽  
Strand Break ◽  
Dna Helicase ◽  
Similar Degree ◽  
Atpase Reaction ◽  
Weak Binding ◽  
Genomic Damage ◽  
Enzymatic Adaptation ◽  
Degree Of Coupling ◽  
Dna Substrate

AbstractDouble strand breaks are the severest genomic damage requiring rapid repair response. In prokaryotes, members of the RecBCD family initiate DNA unwinding essential for double strand break repair mechanism by homologous recombination. RecBCD is a highly processive DNA helicase with an unwinding rate approaching ∼1,600 bp·s-1. The ATPase reaction mechanism enabling RecBCD to achieve this fast unwinding rate and its enzymatic adaptation are not fully understood. Here, we present thermodynamic investigation of DNA and nucleotide binding to RecBCD to reveal the binding linkage and the degree of coupling between its nucleotide cofactor and DNA substrate binding. We find that RecBCD exhibits a weak binding state in the presence of ADP towards double overhang DNA substrate (dohDNA), and the same degree of coupling is observed for RecBCD affinity toward ADP, only in the presence of dohDNA. In the absence of nucleotide cofactor (APO state) or in the presence of AMPpNp, much weaker coupling is observed between the binding of DNA and the nucleotide state towards RecBCD. Other DNA substrates that are not optimally engaged with RecBCD do not exhibit similar degree of coupling. This may be the first evidence for strong and weak binding states that can, in principle, regulate a ‘stepping mechanism’ during processive translocation of RecBCD.

scholarly journals The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

2021 ◽  
Author(s):  
Dipti Vinayak Vernekar ◽  
Giordano Reginato ◽  
Céline Adam ◽  
Lepakshi Ranjha ◽  
Florent Dingli ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Dna Helicase ◽  
Clamp Loader ◽  
Repair Synthesis ◽  
Dna Repair Synthesis ◽  
Gene Conversions

Abstract Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

scholarly journals Large domain movements upon UvrD dimerization and helicase activation

2017 ◽  
Vol 114 (46) ◽  
pp. 12178-12183 ◽  
Author(s):  
Binh Nguyen ◽  
Yerdos Ordabayev ◽  
Joshua E. Sokoloski ◽  
Elizabeth Weiland ◽  
Timothy M. Lohman
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Self Assembly ◽  
Total Internal Reflection ◽  
Dna Helicase ◽  
Conformational State ◽  
Helicase Activity ◽  
Multiple Conformations ◽  
Dna Substrate

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

scholarly journals DDX11 loss causes replication stress and pharmacologically exploitable DNA repair defects

2021 ◽  
Vol 118 (17) ◽  
pp. e2024258118
Author(s):  
Nanda Kumar Jegadesan ◽  
Dana Branzei
Keyword(s):  
Dna Repair ◽  
Cancer Cells ◽  
Drug Hypersensitivity ◽  
Replication Stress ◽  
Strand Break ◽  
Parp Inhibitors ◽  
Dna Helicase ◽  
Brca1 And Brca2 ◽  
Focus Formation ◽  
Chemotherapy Drug

DDX11 encodes an iron–sulfur cluster DNA helicase required for development, mutated, and overexpressed in cancers. Here, we show that loss of DDX11 causes replication stress and sensitizes cancer cells to DNA damaging agents, including poly ADP ribose polymerase (PARP) inhibitors and platinum drugs. We find that DDX11 helicase activity prevents chemotherapy drug hypersensitivity and accumulation of DNA damage. Mechanistically, DDX11 acts downstream of 53BP1 to mediate homology-directed repair and RAD51 focus formation in manners nonredundant with BRCA1 and BRCA2. As a result, DDX11 down-regulation aggravates the chemotherapeutic sensitivity of BRCA1/2-mutated cancers and resensitizes chemotherapy drug–resistant BRCA1/2-mutated cancer cells that regained homologous recombination proficiency. The results further indicate that DDX11 facilitates recombination repair by assisting double strand break resection and the loading of both RPA and RAD51 on single-stranded DNA substrates. We propose DDX11 as a potential target in cancers by creating pharmacologically exploitable DNA repair vulnerabilities.

DOCA-enhanced sites of vasopressin-stimulated cAMP formation in rat cortical collecting tubule

1992 ◽  
Vol 263 (6) ◽  
pp. F1093-F1097
Author(s):  
S. McArdle ◽  
R. Fallet ◽  
W. B. Jeffries ◽  
W. A. Pettinger
Keyword(s):  
Nucleotide Binding ◽  
Similar Degree ◽  
Guanine Nucleotide ◽  
Biochemical Effect ◽  
Collecting Tubule ◽  
Guanine Nucleotide Binding ◽  
And Control ◽  
Gamma S ◽  
Camp Formation ◽  
Cortical Collecting Tubule

In deoxycorticosterone acetate (DOCA)-NaCl hypertension, the effects of vasopressin (VP) in the cortical collecting tubule (CCT) are exaggerated. These include both the biochemical effect of VP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in the CCT and physiological effects of VP-mediated sodium and water retention. In this study, we examined the mechanism of enhanced VP-stimulated cAMP formation in the CCT. We compared cAMP formation in response to activators (following in parentheses) of the VP receptor (VP), of the stimulatory guanine nucleotide binding (Gs) protein [guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S); F-], and of the catalytic subunit of adenylyl cyclase (forskolin, Mn2+) between control and DOCA-NaCl-treated rats. The effects of VP and forskolin were enhanced in CCT of DOCA-NaCl-treated animals by 201 and 139%, respectively, compared with control animals. Other activators, Mn2+ (150%), F- (142%), and GTP gamma S (156%), also caused augmented cAMP formation in the CCT of DOCA-NaCl-treated rats. The DOCA-NaCl-induced increment in cAMP response to VP remained after pretreatment of the rats with pertussis toxin (171 and 169% increase in response in DOCA-NaCl and control rats, respectively), suggesting that altered inhibitory guanine nucleotide binding (Gi) protein function is not the mechanism for the altered response to VP in the CCT. Further evidence that Gi function is intact in DOCA-NaCl animals is that epinephrine (via alpha 2-adrenoceptor stimulation) inhibited VP-stimulated cAMP accumulation to a similar degree in DOCA-NaCl and control rats (86 and 76%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 224 ◽  
Author(s):  
Julius Muellner ◽  
Kristina H. Schmidt
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Strand Break ◽  
Dna Helicase ◽  
Yeast Cells ◽  
Genome Integrity ◽  
Yeast Genome ◽  
Functional Evaluation ◽  
Cellular Functions ◽  
Yeast Homolog

The two PIF1 family helicases in Saccharomyces cerevisiae, Rrm3, and ScPif1, associate with thousands of sites throughout the genome where they perform overlapping and distinct roles in telomere length maintenance, replication through non-histone proteins and G4 structures, lagging strand replication, replication fork convergence, the repair of DNA double-strand break ends, and transposable element mobility. ScPif1 and its fission yeast homolog Pfh1 also localize to mitochondria where they protect mitochondrial genome integrity. In addition to yeast serving as a model system for the rapid functional evaluation of human Pif1 variants, yeast cells lacking Rrm3 have proven useful for elucidating the cellular response to replication fork pausing at endogenous sites. Here, we review the increasingly important cellular functions of the yeast PIF1 helicases in maintaining genome integrity, and highlight recent advances in our understanding of their roles in facilitating fork progression through replisome barriers, their functional interactions with DNA repair, and replication stress response pathways.

scholarly journals Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse

2007 ◽  
Vol 27 (8) ◽  
pp. 2812-2820 ◽  
Author(s):  
Masaoki Kohzaki ◽  
Atsushi Hatanaka ◽  
Eiichiro Sonoda ◽  
Mitsuyoshi Yamazoe ◽  
Koji Kikuchi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Replication Fork ◽  
Strand Break ◽  
Dna Helicase ◽  
Dna Helicases ◽  
Homologous Chromosomes ◽  
Dna Damaging Agents ◽  
Replication Fork Arrest

ABSTRACT Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomyces pombe to chickens and humans. Here, we report the disruption of the FBH1 gene in DT40 cells. Although the yeast fbh1 mutant shows an increase in sensitivity to DNA damaging agents, FBH1 − / − DT40 clones show no prominent sensitivity, suggesting that the loss of FBH1 might be compensated by other genes. However, FBH1 − / − cells exhibit increases in both sister chromatid exchange and the formation of radial structures between homologous chromosomes without showing a defect in homologous recombination. This phenotype is reminiscent of BLM − / − cells and suggests that Fbh1 may be involved in preventing extensive strand exchange during homologous recombination. In addition, disruption of RAD54, a major homologous recombination factor in FBH1 − / − cells, results in a marked increase in chromosome-type breaks (breaks on both sister chromatids at the same place) following replication fork arrest. Further, FBH1 BLM cells showed additive increases in both sister chromatid exchange and the formation of radial chromosomes. These data suggest that Fbh1 acts in parallel with Bloom helicase to control recombination-mediated double-strand-break repair at replication blocks and to reduce the frequency of crossover.

scholarly journals Sgs1 Helicase Activity Is Required for Mitotic but Apparently Not for Meiotic Functions

2000 ◽  
Vol 20 (17) ◽  
pp. 6399-6409 ◽  
Author(s):  
Atsuko Miyajima ◽  
Masayuki Seki ◽  
Fumitoshi Onoda ◽  
Miwa Shiratori ◽  
Nao Odagiri ◽  
...  
Keyword(s):  
Dna Recombination ◽  
Strand Break ◽  
Dna Helicase ◽  
Helicase Activity ◽  
Gene Encoding ◽  
Spore Viability ◽  
The Poor ◽  
High Viability ◽  
Growth Assay ◽  
Break Formation

ABSTRACT The SGS1 gene of Saccharomyces cerevisiaeis a homologue for the Bloom's syndrome and Werner's syndrome genes. The disruption of the SGS1 gene resulted in very poor sporulation, and the majority of the cells were arrested at the mononucleated stage. The recombination frequency measured by a return-to-growth assay was reduced considerably in sgs1disruptants. However, double-strand break formation, which is a key event in the initiation of meiotic DNA recombination, occurred; crossover and noncrossover products were observed in the disruptants, although the amounts of these products were slightly decreased compared with those in wild-type cells. The spores produced by sgs1disruptants showed relatively high viability. The sgs1 spo13 double disruptants sporulated poorly, like thesgs1 disruptants, but spore viability was reduced much more than with either sgs1 or spo13 single disruptants. Disruption of the RED1 or RAD17gene partially alleviated the poor-sporulation phenotype ofsgs1 disruptants, indicating that portions of the population of sgs1 disruptants are blocked by the meiotic checkpoint. The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants.

scholarly journals Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

PLoS ONE ◽  
2012 ◽  
Vol 7 (5) ◽  
pp. e36960 ◽  
Author(s):  
Seetha V. Balasingham ◽  
Ephrem Debebe Zegeye ◽  
Håvard Homberset ◽  
Marie L. Rossi ◽  
Jon K. Laerdahl ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Substrate Specificity ◽  
Dna Helicase ◽  
Enzymatic Activities ◽  
Dna Substrate

scholarly journals YGR042W/MTE1 Functions in Double-Strand Break Repair with MPH1

2015 ◽  
Author(s):  
Askar Yimit ◽  
TaeHyung Kim ◽  
Ranjith Anand ◽  
Sarah Meister ◽  
Jiongwen Ou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Helicase ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Break Repair

Double-strand DNA breaks occur upon exposure of cells to agents such as ionizing radiation and ultraviolet light or indirectly through replication fork collapse at DNA damage sites. If left unrepaired double-strand breaks can cause genome instability and cell death. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination re-localize into discrete nuclear foci. We identified 29 proteins that co-localize with the recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase Mph1 is absent. Mte1 and Mph1 form a complex, and are recruited to double-strand breaks in vivo in a mutually dependent manner. Mte1 is important for resolution of Rad52 foci during double-strand break repair, and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

scholarly journals Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing

2013 ◽  
Vol 42 (4) ◽  
pp. 2380-2390 ◽  
Author(s):  
Shreya Paliwal ◽  
Radhakrishnan Kanagaraj ◽  
Andreas Sturzenegger ◽  
Kamila Burdova ◽  
Pavel Janscak
Keyword(s):  
Molecular Mechanisms ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Inhibitory Effect ◽  
Double Strand Break ◽  
Dna Helicase ◽  
Break Site ◽  
Strand Annealing

Abstract Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.

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