An Endonuclease Activity from
 Escherichia coli
 Absent from Certain
 rec
 -
 Strains

Goldmark, Peter J.; Linn, Stuart

doi:10.1073/pnas.67.1.434

Cited by 151 publications

(47 citation statements)

References 14 publications

(1 reference statement)

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“…The enzyme itself was discovered as a potent exonuclease activity in E. coli (26,113,114,143,177,215,216,282,300,320,321). It is maintained at a low copy number of about 10 RecBCD molecules per cell (82,285), because overproduction actually impairs recombinational DNA repair and increases chromosomal degradation (82).…”

Section: Discoverymentioning

confidence: 99%

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Dillingham

Kowalczykowski

2008

Microbiol Mol Biol Rev

501

599

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SUMMARY The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.

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Section: Discoverymentioning

confidence: 99%

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Dillingham

Kowalczykowski

2008

Microbiol Mol Biol Rev

501

599

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“…The RecB protein associated with RecC has DNA helicase activity but no nuclease activity ( 18 ), while RecD is essential for the nuclease activity ( 17 ). Mutations in either recB, recC or recD result in the loss of an ATP-dependent nuclease activity that is present in wild-type and recA mutant cells ( [19][20][21][22] ). Since plasmid DNA is unstable in recD mutants ( 23 ) and recB mutants do not have the RecBCD ATP-dependent nuclease activity, we chose to introduce a recB insertion mutation (recB268::Tn 10 ) into BWKuLig#1 to test whether RecBCD was generating the deletions in the pBestluc repair products.…”

Section: Dna End-joining Activity Of Paci-linearized Dna In Recb-defimentioning

confidence: 99%

Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology

et al. 2007

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Unlike E. coli, Mycobacterium tuberculosis (Mt) expresses a Ku-like protein and an ATP-dependent DNA ligase that can perform non-homologous end-joining (NHEJ). We have expressed the Mt-Ku and Mt-Ligase D in E. coli using an arabinose-inducible promoter and expression vectors that integrate into specific sites in the E. coli chromosome. E. coli strains have been generated that express the Mt-Ku and Mt-Ligase D on a genetic background that is wild-type for repair, or deficient in either the RecA or RecB protein. Transformation of these strains with linearized plasmid DNA containing a 2-bp overhang has demonstrated that expression of both the Mt-Ku and Mt-Ligase D is required for DNA end-joining and that loss of RecA does not prevent this double-strand break repair. Analysis of the re-joined plasmid has shown that repair is predominantly inaccurate and results in the deletion of sequences. Loss of RecB did not prevent the formation of large deletions, but did increase the amount of end-joining. Sequencing the junctions has revealed that the majority of the ligations occurred at regions of micro-homology (1-4 bps), eliminating one copy of the homologous sequence at the junction. The Mt-Ku and Mt-Ligase D can therefore function in E. coli to re-circularize linear plasmid.

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“…Short periods of heat treatment (low dose) cause local opening of DNA at various points (Inman, 1966). These specific regions are attacked by DNAase(s) (Goldmark & Linn, 1970;Sedgwick & Bridges, 1972) resulting in nicked DNA. A few breaks are repaired by DNA polymerase I and ligase (Town, Smith & Kaplan, 1971Srivastava, 1974), while the remaining breaks require functional lex and recA genes.…”

Section: Discussionmentioning

confidence: 99%

Effect of Incubation Media on the Recovery of Escherichia coli K12 Heated at 52 0C

Ahmad

Srivastava

Agarwala

1978

Journal of General Microbiology

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37The exposure of exponentially grown Escherichia coli ~1 2 to 52 "C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 "C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not.As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 "C, the bacteria regained their resistance to U.V. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of U.V. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.

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An Endonuclease Activity from Escherichia coli Absent from Certain rec ^- Strains

Cited by 151 publications

References 14 publications

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology

Effect of Incubation Media on the Recovery of Escherichia coli K12 Heated at 52 0C

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An Endonuclease Activity from Escherichia coli Absent from Certain rec - Strains

Cited by 151 publications

References 14 publications

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology

Effect of Incubation Media on the Recovery of Escherichia coli K12 Heated at 52 0C

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An Endonuclease Activity from Escherichia coli Absent from Certain rec ^- Strains