scholarly journals The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Gábor M. Harami ◽  
Nikolett T. Nagy ◽  
Máté Martina ◽  
Keir C. Neuman ◽  
Mihály Kovács
Keyword(s):  
Dna Unwinding ◽  
Recq Helicase ◽  
Dna Translocation ◽  
Single Stranded Dna ◽  
E Coli ◽  
Double Stranded Dna

scholarly journals Effects of recJ, recQ, and recFOR Mutations on Recombination in Nuclease-Deficient recB recD Double Mutants of Escherichia coli

2005 ◽  
Vol 187 (4) ◽  
pp. 1350-1356 ◽  
Author(s):  
Ivana Ivančić-Baće ◽  
Erika Salaj-Šmic ◽  
Krunoslav Brčić-Kostić
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Reca Protein ◽  
Recq Helicase ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Recf Pathway ◽  
Recombination Pathway ◽  
Recbcd Enzyme ◽  
Dna Ends

ABSTRACT The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5′→3′ exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5′→3′ exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5′→3′ exonuclease is an element of the RecF recombination machinery.

scholarly journals Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

2015 ◽  
Vol 112 (50) ◽  
pp. E6852-E6861 ◽  
Author(s):  
Behzad Rad ◽  
Anthony L. Forget ◽  
Ronald J. Baskin ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Single Molecule ◽  
Fluorescent Sensor ◽  
Holliday Junctions ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Dna Breaks ◽  
Dna Helicases ◽  
Double Stranded Dna ◽  
Functional Forms ◽  
And Migration

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.

scholarly journals Single-Stranded DNA Translocation of E. coli UvrD Monomer Is Tightly Coupled to ATP Hydrolysis

2012 ◽  
Vol 418 (1-2) ◽  
pp. 32-46 ◽  
Author(s):  
Eric J. Tomko ◽  
Christopher J. Fischer ◽  
Timothy M. Lohman
Keyword(s):  
Atp Hydrolysis ◽  
Dna Translocation ◽  
Single Stranded Dna ◽  
E Coli ◽  
Tightly Coupled

scholarly journals Translocation of E. coli RecQ Helicase on Single-Stranded DNA

Biochemistry ◽  
2012 ◽  
Vol 51 (13) ◽  
pp. 2921-2929 ◽  
Author(s):  
Behzad Rad ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Recq Helicase ◽  
Single Stranded Dna ◽  
E Coli

scholarly journals Defective processing of methylated single-stranded DNA by E. coli alkB mutants

2000 ◽  
Vol 14 (16) ◽  
pp. 2097-2105
Author(s):  
Suneet Dinglay ◽  
Sarah C. Trewick ◽  
Tomas Lindahl ◽  
Barbara Sedgwick
Keyword(s):  
Dna Repair ◽  
Replication Forks ◽  
Human Homolog ◽  
Γ Irradiation ◽  
Single Stranded Dna ◽  
E Coli ◽  
Double Stranded Dna ◽  
The Subject ◽  
M13 Phage ◽  
Methylating Agents

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agentN-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. ArecA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells.

scholarly journals Recombination Hotspots and Single-Stranded DNA Binding Proteins Couple DNA Translocation to DNA Unwinding by the AddAB Helicase-Nuclease

2011 ◽  
Vol 42 (6) ◽  
pp. 806-816 ◽  
Author(s):  
Joseph T.P. Yeeles ◽  
Kara van Aelst ◽  
Mark S. Dillingham ◽  
Fernando Moreno-Herrero
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Dna Unwinding ◽  
Dna Translocation ◽  
Single Stranded Dna ◽  
Recombination Hotspots

scholarly journals sbcB15 and ΔsbcB Mutations Activate Two Types of RecF Recombination Pathways in Escherichia coli

2006 ◽  
Vol 188 (21) ◽  
pp. 7562-7571 ◽  
Author(s):  
Ksenija Zahradka ◽  
Sanela Šimić ◽  
Maja Buljubašić ◽  
Mirjana Petranović ◽  
Damir Đermić ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recq Helicase ◽  
Dna Breaks ◽  
Γ Irradiation ◽  
E Coli ◽  
Exonuclease I ◽  
Double Stranded Dna ◽  
Recombination Processes ◽  
Recf Pathway ◽  
Recombination Reactions

ABSTRACT Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and ΔsbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and ΔsbcB mutations on DNA repair after UV and γ irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than ΔsbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as ΔsbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC ΔsbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 595-605 ◽  
Author(s):  
Bradley J Merrill ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Synthetic Lethality ◽  
Velocity Sedimentation ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Dna Breaks ◽  
Single Stranded Dna ◽  
Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

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