RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro.

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.

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Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

10.1101/2021.07.18.452824 ◽

2021 ◽

Author(s):

Kazuto Yoshimi ◽

Kohei TAKESHITA ◽

Noriyuki Kodera ◽

Satomi Shibumura ◽

Yuko Yamauchi ◽

...

Keyword(s):

Escherichia Coli ◽

High Speed ◽

Dna Helicase ◽

Type I ◽

Loop Formation ◽

Force Microscopy ◽

Atomic Force ◽

Target Strand ◽

Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

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DNA Helicase Activity of PcrA Is Not Required for the Displacement of RecA Protein from DNA or Inhibition of RecA-Mediated Strand Exchange

Journal of Bacteriology ◽

10.1128/jb.00376-07 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4502-4509 ◽

Cited By ~ 23

Author(s):

Syam P. Anand ◽

Haocheng Zheng ◽

Piero R. Bianco ◽

Sanford H. Leuba ◽

Saleem A. Khan

Keyword(s):

Dna Recombination ◽

Reca Protein ◽

Resonance Energy ◽

Dna Helicase ◽

Strand Exchange ◽

Single Pair ◽

Helicase Activity ◽

Dna Strand Exchange ◽

Dna Strand

ABSTRACT PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

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Characterization of the Helicase Activity and Substrate Specificity of Mycobacterium tuberculosis UvrD

Journal of Bacteriology ◽

10.1128/jb.01421-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1542-1555 ◽

Cited By ~ 28

Author(s):

Elena Curti ◽

Stephen J. Smerdon ◽

Elaine O. Davis

Keyword(s):

Mycobacterium Tuberculosis ◽

Atpase Activity ◽

Amino Acid Identity ◽

Dna Helicase ◽

Helicase Activity ◽

Dna Oligonucleotides ◽

Promising Target ◽

Sites Of Action

ABSTRACT UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3′-single-stranded tailed duplex substrates over 5′-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3′-to-5′ polarity. A 3′ single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.

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Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)50649-4 ◽

1992 ◽

Vol 267 (6) ◽

pp. 4207-4214

Author(s):

L.J. Roman ◽

A.K. Eggleston ◽

S.C. Kowalczykowski

Keyword(s):

Escherichia Coli ◽

Dna Helicase ◽

Helicase Activity ◽

Recbcd Enzyme

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Different Phenotypesin VivoAre Associated With ATPase Motif Mutations inSchizosaccharomyces pombeMinichromosome Maintenance Proteins

Genetics ◽

10.1093/genetics/160.4.1305 ◽

2002 ◽

Vol 160 (4) ◽

pp. 1305-1318 ◽

Cited By ~ 3

Author(s):

Eliana B Gómez ◽

Michael G Catlett ◽

Susan L Forsburg

Keyword(s):

Fission Yeast ◽

Protein Function ◽

Dna Helicase ◽

Helicase Activity ◽

Mcm Proteins ◽

Atp Binding And Hydrolysis ◽

In Vivo Effects ◽

Aaa Atpases

AbstractThe six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA+ ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.

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UvrD helicase activation by MutL involves rotation of its 2B subdomain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905513116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16320-16325 ◽

Cited By ~ 7

Author(s):

Yerdos A. Ordabayev ◽

Binh Nguyen ◽

Alexander G. Kozlov ◽

Haifeng Jia ◽

Timothy M. Lohman

Keyword(s):

Escherichia Coli ◽

Single Molecule ◽

Self Assembly ◽

Kinetic Studies ◽

Conformational State ◽

Helicase Activity ◽

Single Molecule Fret ◽

Open Conformation ◽

Dna Complex

Escherichia coli UvrD is a superfamily 1 helicase/translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an Escherichia coli MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer–DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain.

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Saccharomyces cerevisiae Srs2 DNA Helicase Selectively Blocks Expansions of Trinucleotide Repeats

Molecular and Cellular Biology ◽

10.1128/mcb.24.17.7324-7330.2004 ◽

2004 ◽

Vol 24 (17) ◽

pp. 7324-7330 ◽

Cited By ~ 63

Author(s):

Saumitri Bhattacharyya ◽

Robert S. Lahue

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Recombination ◽

Dna Helicase ◽

Trinucleotide Repeats ◽

Model Organisms ◽

Helicase Activity ◽

Dna Polymerase Delta ◽

Dinucleotide Repeats ◽

Cgg Repeats

ABSTRACT Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinucleotide repeats, at unique sequences, or for TNR contractions in srs2 mutants were not altered. Srs2 is known to suppress inappropriate genetic recombination; however, the TNR expansion phenotype of srs2 mutants was largely independent of RAD51 and RAD52. Instead, Srs2 mainly functioned with DNA polymerase delta to block expansions. The helicase activity of Srs2 was important, because a point mutant lacking ATPase function was defective in blocking expansions. Purified Srs2 was substantially better than bacterial UvrD helicase at in vitro unwinding of a DNA substrate that mimicked a TNR hairpin. Disruption of the related helicase gene SGS1 did not lead to excess expansions, nor did wild-type SGS1 suppress the expansion phenotype of an srs2 strain. We conclude that Srs2 selectively blocks triplet repeat expansions through its helicase activity and primarily in conjunction with polymerase delta.

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