Quantitation of the Involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC Genes in the Repair of X-Ray-Induced DNA Double-Strand Breaks in Escherichia coli

Sargentini, Neil J.; Smith, Kendric C.

doi:10.2307/3576850

Cited by 130 publications

(81 citation statements)

References 45 publications

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“…The efficiency of DSB repair in wild-type cells is dramatically reduced by recN mutations (Picksley et al, 1984;Sargentini and Smith, 1986). Consistent with our proposal that RDR is involved in DSB repair, we have found that a recN mutation greatly reduces RDR in rec + sbc + cells.…”

Section: Possible Roles Of Rdr In Dsb Repairsupporting

confidence: 91%

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Asai

Bates

Kogoma

1994

Cell

113

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SummaryHomologous recombination-dependent DNA replication (RDR) of a λ cos site-carrying plasmid is demonstrated in E. coli cells when the cells express λ terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot χ. χ is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the θ mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (or/Ms) on the chromosome.

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Section: Possible Roles Of Rdr In Dsb Repairsupporting

confidence: 91%

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Asai

Bates

Kogoma

1994

Cell

113

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“…We propose that the reason for this is the inefficient repair of double-strand breaks in strains deficient for recB, recC, or both (34 (48) and that recD cells are even more resistant than wild-type cells to gamma irradiation (21) are in accord with the proposal that recBC-dependent unwinding of duplex DNA ends initiates efficient recombination repair at doublestrand breaks.…”

Section: Discussionmentioning

confidence: 69%

Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding

1992

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Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49%6 of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% ofthe DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) orxonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recCf mutant (in which the RecD protein is intact but does not function) and its recJ, xonA4, and recJxonA4 derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.Apart from RecA protein, the RecBCD enzyme is the principal component of the major route of homologous recombination in Escherichia coli (5,19,37). In vitro, the enzyme has multiple enzymatic activities, including ATPdependent exonuclease for double-and single-stranded DNA, ATP-stimulated endonuclease for single-stranded DNA, and ATP-dependent DNA-unwinding activity (40, 42). The recB, recC, and recD genes code for the three subunits of the RecBCD enzyme in E. coli (1, 2, 6) and other bacteria (22,28,50,51 characteristic of the RecBCD enzyme was an enigma (36,40).We studied the interaction of RecBCD and RecBC enzymes with linear duplex DNA in vivo. We used infection of cells with a phage T4 gene 2am mutant (T4 2-) to transport labeled linear duplex DNA into cells and followed its degradation. Phage T4 2-lacks the gene 2 pilot protein on its DNA ends. This protein protects the phage genome from degradation by the RecBCD enzyme (24). MATERIALS AND METHODSBacterial strains. The strains employed were mostly E. coli AB1157 and its derivatives ( Table 1). The AB1157 genetic background was chosen because of its supE44 suppressor of nonsense mutations. First, it suppresses the gene 2 amber mutation of T4 2-, allowing undegraded genomes to be equipped with the gene 2 proteins necessary for subsequent successful infection of a recB+ C+ D+ cell. Second, the suppressor does not suppress the nonsense allele recDlOll (1) used in this study. The bacterial strains were constructed by P1 transduction. The selection for the recJ284::TnlO allele (15) wa...

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“…Unlike other members of the RecF pathway, recN mutants are quite sensitive to ionizing radiation or mitomycin C, and the repair of multiple, site-specific DSBs does not occur in the absence of RecN (8,12,13). The recN gene is also required for the suppression of chromosomal rearrangements and deletions (12) during the repair of a single DSB.…”

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confidence: 99%

RecN Is a Cohesin-like Protein That Stimulates Intermolecular DNA Interactions in Vitro

Reyes

Patidar

Uranga

et al. 2010

Journal of Biological Chemistry

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The bacterial RecN protein is involved in the recombinational repair of DNA double-stranded breaks, and recN mutants are sensitive to DNA-damaging agents. Little is known about the biochemical function of RecN. Protein sequence analysis suggests that RecN is related to the SMC (structural maintenance of chromosomes) family of proteins, predicting globular N-and C-terminal domains connected by an extensive coil-coiled domain. The N-and C-domains contain the nucleotide-binding sequences Walker A and Walker B, respectively. We have purified the RecN protein from Deinococcus radiodurans and characterized its DNA-dependent and DNA-independent ATPase activity. The cellular genome maintenance processes of DNA replication, recombination, and repair are highly interconnected, sharing multiple pathways and common enzymes. A physical understanding of the function of key enzymes involved in genome maintenance processes is critical for elucidating the basic DNA metabolic strategies of cells. The primary function of homologous DNA recombination in mitotic cells as well as bacterial cells is the nonmutagenic repair of replication forks that have stalled or collapsed at noncoding lesions or breaks (1-3). When a replication fork encounters a break in the phosphodiester backbone of one template strand, a double-stranded break (DSB) 2 results (4). Recombinational DSB repair pathways promote strand invasion to regenerate a fork structure. In Escherichia coli, these pathways include the helicase and nuclease functions of the RecBCD complex, and the RecA protein. RecN, among various other proteins, is required for DSB repair in bacteria. Existing DSB repair models do not encompass RecN, because no clear function has been ascribed to the protein as yet.The recN gene of E. coli (also known as radB (5)) was identified by two groups over 20 years ago. Lloyd et al. (6) isolated a mutation (rec-259) that caused increased sensitivity to UV light in a recBC sbcB background. Independently, Sargentini and Smith (7) isolated the radB101 mutation, which caused sensitivity to ␥-irradiation and mitomycin C. Expression of the recN gene is regulated by the LexA repressor (8, 9), and the RecN protein is one of the most abundantly produced proteins induced as part of the SOS response to DNA damage (10). Once produced in response to damage, RecN protein has a very short half-life (ϳ10 min), because it apparently is rapidly proteolyzed by the ClpXP protease system (11).The recN gene product was originally placed in the RecF-dependent gap repair pathway, but substantial evidence suggests that RecN is also involved in the RecBCD DSB repair pathway. Unlike other members of the RecF pathway, recN mutants are quite sensitive to ionizing radiation or mitomycin C, and the repair of multiple, site-specific DSBs does not occur in the absence of RecN (8,12,13). The recN gene is also required for the suppression of chromosomal rearrangements and deletions (12) during the repair of a single DSB. Given this wealth of genetic data, the RecN protein is clearly involve...

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Quantitation of the Involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC Genes in the Repair of X-Ray-Induced DNA Double-Strand Breaks in Escherichia coli

Cited by 130 publications

References 45 publications

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding

RecN Is a Cohesin-like Protein That Stimulates Intermolecular DNA Interactions in Vitro

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