The effect of lexA and recF mutations on post-replication repair and DNA synthesis in Escherichia coli K-12

Ganesan, Ann K.; Seawell, Patricia C.

doi:10.1007/bf00341799

Cited by 93 publications

(53 citation statements)

References 24 publications

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“…Cultures were then placed into nonradioactive media, and at the indicated times, cells were lysed and the size of the labeled DNA fragments was analyzed on alkaline sucrose gradients. Whereas the pulse-labeled 3 H-DNA cosedimented with the large 14 C-labeled genomic DNA in mock-irradiated cultures (data not shown), the DNA synthesized immediately after irradiation consisted of smaller, slower-migrating fragments in UV-irradiated cultures (Fig. 4).…”

Section: Resultsmentioning

confidence: 85%

“…Consistent with the gapped products observed on plasmids, a number of previous studies have used alkali sucrose gradient analysis to show that immediately after UV irradiation, a limited amount of DNA synthesis can be detected on the chromosome that also contains gaps (13,42,43). The repair (or joining) of the chromosomal gapped DNA fragments appears to depend on at least some of the same gene products that are required for the resumption of DNA synthesis, including RecA, RecF, RecO, and RecR (14,41,47). Several early studies characterizing the processing and repair of nascent-strand gaps were performed prior to the discovery of the concept that translesion synthesis or nucleotide excision repair may be involved in this pathway.…”

Section: Resultsmentioning

confidence: 99%

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Nucleotide Excision Repair or Polymerase V-Mediated Lesion Bypass Can Act To Restore UV-Arrested Replication Forks in Escherichia coli

2005

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Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.Irradiation of cells with 254-nm UV light induces lesions that block DNA polymerases. Lesions that block polymerases are thought to either arrest the progress of the replication machinery or produce nascent-strand gaps depending on which template strand contains the lesion (3,4,17,32,45,50,53,54). Several studies using plasmid substrates indicate that lesions in the leading-strand template arrest the overall progression of the replication fork, with the nascent lagging strand continuing a short distance beyond the arrested leading strand (17,30,50,53). In contrast, lesions in the lagging-strand template are thought to generate gaps in the nascent DNA strand at sites opposite to the lesion, presumably because discontinuous synthesis of the lagging strand allows the blocked polymerase to reinitiate downstream of the lesion site (17,30,50). Events that are consistent with this can also be seen on the chromosome of UV-irradiated Escherichia coli. Following a moderate dose of UV irradiation, the rate of DNA synthesis is transiently inhibited before it efficiently recovers at a time that correlates with lesion removal (8,45). During this period of inhibition, some limited DNA synthesis is still observed that contains gaps, consistent with replication continuing past a subset of the lesions in the template (13,42,43). The repair and restoration of the DNA template in each of these two situations may involve unique enzymatic pathways and are likely to have different consequences for the cell with respect to survival and mutagenesis.Lesions that arrest the overall progression of the replication machinery would be expected to prevent the replication of the genome and are likely to result in cell lethality if the block to replication cannot be overcome. The a...

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Nucleotide Excision Repair or Polymerase V-Mediated Lesion Bypass Can Act To Restore UV-Arrested Replication Forks in Escherichia coli

2005

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“…Several studies, including our own, have linked the synergism between recA function and excision repair, to an effect on the recovery of replication following UV irradiation (21,(45)(46)(47). Furthermore, other studies have observed a strong correlation between the time at which replication recovers and the time at which DNA repair is almost complete (45,48,49).…”

Section: A Common Pathway Involving Both Nucleotide Excision Repair Amentioning

confidence: 79%

Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated Escherichia coli need not involve recombination

Courcelle

Hanawalt

2001

Proc. Natl. Acad. Sci. U.S.A.

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Alternative reproductive cycles make use of different strategies to generate different reproductive products. In Escherichia coli, recA and several other rec genes are required for the generation of recombinant genomes during Hfr conjugation. During normal asexual reproduction, many of these same genes are needed to generate clonal products from UV-irradiated cells. However, unlike conjugation, this latter process also requires the function of the nucleotide excision repair genes. Following UV irradiation, the recovery of DNA replication requires uvrA and uvrC, as well as recA, recF, and recR. The rec genes appear to be required to protect and maintain replication forks that are arrested at DNA lesions, based on the extensive degradation of the nascent DNA that occurs in their absence. The products of the recJ and recQ genes process the blocked replication forks before the resumption of replication and may affect the fidelity of the recovery process. We discuss a model in which several rec gene products process replication forks arrested by DNA damage to facilitate the repair of the blocking DNA lesions by nucleotide excision repair, thereby allowing processive replication to resume with no need for strand exchanges or recombination. The poor survival of cellular populations that depend on recombinational pathways (compared with that in their excision repair proficient counterparts) suggests that at least some of the rec genes may be designed to function together with nucleotide excision repair in a common and predominant pathway by which cells faithfully recover replication and survive following UV-induced DNA damage.

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“…Genetic data as well as the results of Southern blotting with the cloned dcm gene as a probe suggest that this strain is completely missing the dcm gene (2,4). Thus, these data suggest either that there are additional types of lethal damage caused by 5-azaC (16,33). In addition, the ability of the RecA protein to mediate homologous recombination appears to be more important in this kind of repair than is its ability to induce the SOS response (45).…”

Section: Methodsmentioning

confidence: 99%

Genetic analysis of the 5-azacytidine sensitivity of Escherichia coli K-12

Bhagwat¹,

Roberts²

1987

J Bacteriol

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DNA containing 5-azacytidine (5-azaC) has been shown to form stable detergent-resistant complexes with cytosine methylases. We reasoned that if 5-azaC treatment causes protein-DNA cross-links in vivo, then mutations in DNA repair and recombination genes may increase the sensitivity of a cell to 5-azaC. We found that although recA (defective) and lexA (induction-negative) mutants of Escherichia coli were very sensitive to the drug, mutations in uvrA and ung genes had little effect on cell lethality. The sensitivity of recA strains to 5-azaC was dose dependent and was enhanced by the overproduction of a DNA cytosine methylase in the cell. Unexpectedly, a strain of E. coli carrying a recA mutation and a deletion of the DNA cytosine methylase gene (dcm) was found to be significantly sensitive to 5-azaC. Study of mutations in the pyrimidine salvage pathway of E. coli suggests that direct phosphorylation of 5-azaC, rather than phosphorylation of its degradation products, is largely responsible for the lethal effects of the drug. The addition of uracil to the growth medium had little effect on cell lethality of recA mutants, but it partially reversed the inhibition of cell growth caused by 5-azaC. This reversal of the bacteriostatic effects of the drug could not be achieved by adding cytosine or orotic acid to the growth medium and required the presence of functional UMP-pyrophosphorylase (gene upp) in the cell.5-Azacytidine (5-azaC), an analog of cytidine in which the C-H group at position 5 is replaced by a nitrogen, has a multitude of biological effects in a wide variety of organisms. These effects range from mutagenesis and inhibition of cell growth for bacteria to increase in sister chromatid exchange in hamster cells, as well as induction of differentiation in mouse cells and induction of synthesis of proteins such as hypoxanthine-guanine phosphoribosyl transferase and fetal globin in human cells. At the biochemical level, 5-azaC has been shown to cause hypomethylation of DNA, to produce defective rRNAs and tRNAs, and to inhibit protein synthesis (for recent reviews, see references 20-22, 38, and 39). The ability of 5-azaC to cause hypomethylation of DNA is believed to be the cause of many of the phenotypic effects of the drug (20)(21)(22).The observation by Friedman (12) that a number of bacterial DNA cytosine methylases are inactivated by incubation with DNA containing 5-azaC was the first clue to the mechanism by which 5-azaC causes hypomethylation. On the basis of the results of that study and others, Santi et al. (34) suggested that 5-azaC acts as a mechanism-based inhibitor of cytosine methylases. Specifically, it was suggested that most cytosine methylases link covalently to position 6 of cytosine as a normal intermediate in their action. After the transfer of a methyl group from S-adenosylmethionine to position 5 of cytosine, the enzyme unlinks itself. 5-azaC, when incorporated into DNA or RNA at the site of methylation, sabotages this reaction by making the transfer of the methyl group to position 5 impo...

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The effect of lexA and recF mutations on post-replication repair and DNA synthesis in Escherichia coli K-12

Cited by 93 publications

References 24 publications

Nucleotide Excision Repair or Polymerase V-Mediated Lesion Bypass Can Act To Restore UV-Arrested Replication Forks in Escherichia coli

Nucleotide Excision Repair or Polymerase V-Mediated Lesion Bypass Can Act To Restore UV-Arrested Replication Forks in Escherichia coli

Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated Escherichia coli need not involve recombination

Genetic analysis of the 5-azacytidine sensitivity of Escherichia coli K-12

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