Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli.

Lovett, Susan; Kolodner, Richard D.

doi:10.1073/pnas.86.8.2627

Cited by 257 publications

(193 citation statements)

References 30 publications

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“…The plasmid was used to cover DrecG in a strain also deleted for the lac operon and carrying additional mutations inactivating one or more of the enzymes with known ssDNA exonuclease activity. We tested ExoI (encoded by xonA), which attacks 39 ends (Lehman and Nussbaum 1964); RecJ, which attacks 59 ends (Lovett and Kolodner 1989); and ExoVII (encoded by xseA), which can target either end (Chase and Richardson 1974). We also tested the SbcCD enzyme, which has multiple nuclease activities, including the ability to remove 39 overhangs from partial duplexes and to cut hairpin structures (Chalker et al 1988;Connelly et al 1998Connelly et al , 1999Eykelenboom et al 2008).…”

Section: Resultsmentioning

confidence: 99%

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

et al. 2010

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Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 39 flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that, to stay alive, cells must have either RecG or a 39 single-stranded DNA (ssDNA) exonuclease, which can be exonuclease I, exonuclease VII, or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.

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Section: Resultsmentioning

confidence: 99%

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

et al. 2010

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“…Cells with nonfunctional recD have normal or elevated levels of recombination that is dependent on recJ function (Lovett et al 1988). The RecJ protein is a 5 0 →3 0 single-strand specific exonuclease (Lovett & Kolodner 1989). Thus, the consequence of RecBC enzyme helicase and RecJ protein nuclease action on a dsDNA end would be to produce a 3 0 -ssDNA overhang.…”

Section: Discussionmentioning

confidence: 99%

Chi‐activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit

1997

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Background: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (x) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of x causes both attenuation of the 3 0 →5 0 exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5 0 →3 0 polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that x-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit.

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“…The strain is also named RDK1896 (27). Plasmid pT7POL26 codes for T7 RNA polymerase under the control of a lac promoter (28).…”

Section: Methodsmentioning

confidence: 99%

C-terminal Deletions of the Escherichia coli RecA Protein

Lusetti

Wood

Fleming

et al. 2003

Journal of Biological Chemistry

101

103

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A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK a of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.

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Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli.

Cited by 257 publications

References 30 publications

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

Chi‐activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit

C-terminal Deletions of the Escherichia coli RecA Protein

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