DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli

ABSTRACT Escherichia coli initiates the SOS response when single-stranded DNA (ssDNA) produced by DNA damage is bound by RecA and forms a RecA-DNA filament. recA SOS constitutive [recA(Con)] mutants induce the SOS response in the absence of DNA damage. It has been proposed that recA(Con) mutants bind to ssDNA at replication forks, although the specific mechanism is unknown. Previously, it had been shown that recA4142(F217Y), a novel recA(Con) mutant, was dependent on RecBCD for its high SOS constitutive [SOS(Con)] expression. This was presumably because RecA4142 was loaded at a double-strand end (DSE) of DNA. Herein, it is shown that recA4142 SOS(Con) expression is additionally dependent on ruvAB (replication fork reversal [RFR] activity only) and recJ (5′→3′ exonuclease), xonA (3′→5′ exonuclease) and partially dependent on recQ (helicase). Lastly, sbcCD mutations (Mre11/Rad50 homolog) in recA4142 strains caused full SOS(Con) expression in an ruvAB-, recBCD-, recJ-, and xonA-independent manner. It is hypothesized that RuvAB catalyzes RFR, RecJ and XonA blunt the DSE (created by the RFR), and then RecBCD loads RecA4142 onto this end to produce SOS(Con) expression. In sbcCD mutants, RecA4142 can bind other DNA substrates by itself that are normally degraded by the SbcCD nuclease.

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Events associated with DNA replication disruption are not observed in hydrogen peroxide-treated Escherichia coli

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab044 ◽

2021 ◽

Vol 11 (4) ◽

Author(s):

Chettar A Hoff ◽

Sierra S Schmidt ◽

Brandy J Hackert ◽

Travis K Worley ◽

Justin Courcelle ◽

...

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Dna Damage ◽

Replication Fork ◽

Dna Lesions ◽

Pyrimidine Dimers ◽

Oxygen Sensitive ◽

Recf Pathway ◽

Similar Processing

Abstract UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.

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Answering the Call: Coping with DNA Damage at the Most Inopportune Time

Journal of Biomedicine and Biotechnology ◽

10.1155/s1110724302202016 ◽

2002 ◽

Vol 2 (2) ◽

pp. 66-74 ◽

Cited By ~ 14

Author(s):

David J. Crowley ◽

Justin Courcelle

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Replication Fork ◽

Sos Response ◽

Dna Lesions ◽

Dna Damage Checkpoint ◽

Chromosomal Replication ◽

Replication Process ◽

Comprehensive Picture ◽

High Possibility

DNA damage incurred during the process of chromosomal replication has a particularly high possibility of resulting in mutagenesis or lethality for the cell. The SOS response ofEscherichia coliappears to be well adapted for this particular situation and involves the coordinated up-regulation of genes whose products center upon the tasks of maintaining the integrity of the replication fork when it encounters DNA damage, delaying the replication process (a DNA damage checkpoint), repairing the DNA lesions or allowing replication to occur over these DNA lesions, and then restoring processive replication before the SOS response itself is turned off. Recent advances in the fields of genomics and biochemistry has given a much more comprehensive picture of the timing and coordination of events which allow cells to deal with potentially lethal or mutagenic DNA lesions at the time of chromosomal replication.

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Isolation of SOS Constitutive Mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.186.21.7149-7160.2004 ◽

2004 ◽

Vol 186 (21) ◽

pp. 7149-7160 ◽

Cited By ~ 62

Author(s):

Erin K. O'Reilly ◽

Kenneth N. Kreuzer

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Replication Fork ◽

Reca Protein ◽

Open Reading Frames ◽

Reporter Construct ◽

Mutant Strains ◽

Exogenous Agents ◽

Transposon Insertions ◽

Reading Frames

ABSTRACT The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.

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