Lack of Replication Disruption Following H2O2-induced Damage in \u3ci\u3eEscherichia coli\u3c/i\u3e

Hydrogen peroxide (H2O2) toxicity has long been thought to be predominantly due to oxidative DNA damage that can disrupt DNA replication and result in lethality. Curiously and contrary to this view, it is also well established that the glycosylases responsible for repairing oxidized-base damage are as resistant as wild-type cells when treated with H2O2. The observation raises the possibility that H2O2-induced DNA damage does not disrupt or prevent replication. To examine this possibility, I examined the sensitivity of recF mutants to H2O2. RecF is known to be required to maintain and restore replication forks after disruption by DNA damage. Survival curves of mutants treated with either UV irradiation or H2O2 were generated and, as expected, recF mutants were shown to die off quicker after UV exposure, relative to wild-type cells. However, recF mutants were not hypersensitive to H2O2. The results would be consistent with the idea that DNA damage induced by H2O2 does not disrupt DNA replication and may not factor significantly into its lethality

Inefficient Replication Reduces RecA-mediated Repair of UVdamaged Plasmids introduced into competent \u3ci\u3eEscherichia coli\u3c/i\u3e

Transformation of Escherichia coli with purified plasmids containing DNA damage is frequently used as a tool to characterize repair pathways that operate on chromosomes. In this study, we used an assay that allowed us to quantify plasmid survival and to compare how efficiently various repair pathways operate on plasmid DNA introduced into cells relative to their efficiency on chromosomal DNA. We observed distinct differences between the mechanisms operating on the transforming plasmid DNA and the chromosome. An average of one UV-induced lesion was sufficient to inactivate ColE1-based plasmids introduced into nucleotide excision repair mutants, suggesting an essential role for repair on newly introduced plasmid DNA. By contrast, the absence of RecA, RecF, RecBC, RecG, or RuvAB had a minimal effect on the survival of the transforming plasmid DNA containing UV-induced damage. Neither the presence of an endogenous homologous plasmid nor the induction of the SOS response enhanced the survival of transforming plasmids. Using two-dimensional agarose-gel analysis, both replication- and RecA-dependent structures that were observed on established, endogenous plasmids following UV-irradiation, failed to form on UV-irradiated plasmids introduced into E. coli. We interpret these observations to suggest that the lack of RecA-mediated survival is likely to be due to inefficient replication that occurs when plasmids are initially introduced into cells, rather than to the plasmid’s size, the absence of homologous sequences, or levels of recA expression

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

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 of Escherichia coli appears 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

chi sequences switch the RecBCD helicase–nuclease complex from degradative to replicative modes during the completion of DNA replication

Accurately completing DNA replication when two forks converge is essential to genomic stability. The RecBCD helicase–nuclease complex plays a central role in completion by promoting resection and joining of the excess DNA created when replisomes converge. chi sequences alter RecBCD activity and localize with crossover hotspots during sexual events in bacteria, yet their functional role during chromosome replication remains unknown. Here, we use two-dimensional agarose gel analysis to show that chi induces replication on substrates containing convergent forks. The induced replication is processive but uncoupled with respect to leading and lagging strand synthesis and can be suppressed by ter sites which limit replisome progression. Our observations demonstrate that convergent replisomes create a substrate that is processed by RecBCD and that chi, when encountered, switches RecBCD from a degradative to replicative function. We propose that chi serves to functionally differentiate DNA ends created during completion, which require degradation, from those created by chromosomal double-strand breaks, which require resynthesis

UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair

RecG directs DNA synthesis during double-strand break repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability

Escherichia Coli Fpg Glycosylase is Nonrendundant and Required for the Rapid Global Repair of Oxidized Purine and Pyrimidine Damage In Vivo

Endonuclease (Endo) III and formamidopyrimidine-N-glycosylase (Fpg) are two of the predominant DNA glycosylases in Escherichia coli that remove oxidative base damage. In cell extracts and purified form, Endo III is generally more active toward oxidized pyrimidines, while Fpg is more active towards oxidized purines. However, the substrate specificities of these enzymes partially overlap in vitro. Less is known about the relative contribution of these enzymes in restoring the genomic template following oxidative damage. In this study, we examined how efficiently Endo III and Fpg repair their oxidative substrates in vivo following treatment with hydrogen peroxide. We found that Fpg was nonredundant and required to rapidly remove its substrate lesions on the chromosome. In addition, Fpg also repaired a significant portion of the lesions recognized by Endo III, suggesting that it plays a prominent role in the global repair of both purine damage and pyrimidine damage in vivo. By comparison, Endo III did not affect the repair rate of Fpg substrates and was only responsible for repairing a subset of its own substrate lesions in vivo. The absence of Endo VIII or nucleotide excision repair did not significantly affect the global repair of either Fpg or Endo III substrates in vivo. Surprisingly, replication recovered after oxidative DNA damage in all mutants examined, even when lesions persisted in the DNA, suggesting the presence of an efficient mechanism to process or overcome oxidative damage encountered during replication

Interstrand Crosslink Resistance in Escherichia Coli

8-methoxypsoralen is a photoactivated DNA-intercalating agent, which after absorbing two high-energy UVA photons, covalently binds pyrimidine bases on both DNA strands, to form an interstrand crosslink (ICL). These lesions completely block replication and transcription, and are widely used in chemotherapies; yet the mechanism by which they are processed remains poorly understood. In 1985, Ahmed and Holland reported an Escherichia coli mutant demonstrating hyper-resistance to ICL-inducing agents. The mutation was mapped to 57.2 minutes on the chromosome, and potentially encoded a 55-kDa protein that was induced as part of the SOS response. Although these genes remain unidentified, hscA and hscB map to this location, have a similar size, and are SOS-inducible. To determine if these genes or others might confer ICL resistance in E. coli, we characterized how cells survived psoralen-UVA (PUVA) treatment in the absence of HscAB, and when these gene products were overexpressed. In a second approach to screen for ICL resistance genes, we developed a selection system to isolate hyper-resistant strains through the sequential growth and exposure of wild-type cultures to PUVA. We found no effect on cell survival in the hscAB mutant compared to its wild-type parent, suggesting that HscAB may not be contributing to ICL resistance as previously hypothesized. However, due to the significant cytotoxicity of plasmids containing hscAB, even in the absence of PUVA treatment, we were unable to determine whether over-expression of these gene products might provide a protective effect to cells. Using iteratively PUVA cells, we isolated strains that were \u3e10^4-fold more resistant to this ICL-inducing agent compared to the parent strain. This result suggests that E. coli possess mechanisms to repair or tolerate ICL’s that contribute to resistance to these agents, similar to what is observed in human cancer cells

RecBCD is required to Complete Chromosomal Replication: Implications for Double- Strand Break Frequencies and Repair Mechanisms

Several aspects of the mechanism of homologous double strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process- after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double strand breaks and replication fork collapse occur far less frequently than previously speculated

Cho Endonuclease Functions During DNA Interstrand Crosslink Repair in \u3ci\u3eEscherichia coli\u3c/i\u3e

DNA interstrand crosslinks are complex lesions that covalently link both strands of the duplex DNA. Lesion removal is proposed to initiate via the UvrABC nucleotide excision repair complex, however less is known about the subsequent steps of this complex repair pathway. In this study, we characterized the contribution of nucleotide excision repair mutants to survival in the presence of psoralen-induced damage. Unexpectedly, we observed that the nucleotide excision repair mutants exhibit differential sensitivity to psoralen-induced damage, with uvrC mutants being less sensitive than either uvrA or uvrB. We show that Cho, an alternative endonuclease, acts with UvrAB and is responsible for the reduced hypersensitivity of uvrC mutants. We find that Cho\u27s contribution to survival correlates with the presence of DNA interstrand crosslinks, rather than monoadducts, and operates at a step after, or independent from, the initial incision during the global repair of psoralen DNA adducts from the genome