Manganese transporters regulate the resumption of replication in hydrogen peroxide-stressed Escherichia coli.

Following hydrogen peroxide treatment, ferrous iron (Fe2+) is oxidized to its ferric form (Fe3+), stripping it from and inactivating iron-containing proteins. Many mononuclear iron enzymes can be remetallated by manganese to restore function, while other enzymes specifically utilize manganese as a cofactor, having redundant activities that compensate for iron-depleted counterparts. DNA replication relies on one or more iron-dependent protein(s) as synthesis abates in the presence of hydrogen peroxide and requires manganese in the medium to resume. Here, we show that manganese transporters regulate the ability to resume replication following oxidative challenge in Escherichia coli. The absence of the primary manganese importer, MntH, impairs the ability to resume replication; whereas deleting the manganese exporter, MntP, or transporter regulator, MntR, dramatically increases the rate of recovery. Unregulated manganese import promoted recovery even in the absence of Fur, which maintains iron homeostasis. Similarly, replication was not restored in oxyR mutants, which cannot upregulate manganese import following hydrogen peroxide stress. Taken together, the results define a central role for manganese transport in restoring replication following oxidative stress.

Mutations in AcrR and RNA Polymerase Confer High-Level Resistance to Psoralen-UVA Irradiation.

DNA interstrand cross-links, such as those formed by psoralen-UVA irradiation, are highly toxic lesions in both humans and bacteria, with a single lesion being lethal in Escherichia coli. Despite the lack of effective repair, human cancers and bacteria can develop resistance to cross-linking treatments, although the mechanisms of resistance remain poorly defined. Here, we subjected E. coli to repeated psoralen-UVA exposure to isolate three independently derived strains that were >10,000-fold more resistant to this treatment than the parental strain. Analysis of these strains identified gain-of-function mutations in the transcriptional regulator AcrR and the alpha subunit of RNA polymerase that together could account for the resistance of these strains. Resistance conferred by the AcrR mutation is mediated at least in part through the regulation of the AcrAB-TolC efflux pump. Resistance via mutations in the alpha subunit of RNA polymerase occurs through a still-uncharacterized mechanism that has an additive effect with mutations in AcrR. Both acrR and rpoA mutations reduced cross-link formation in vivo. We discuss potential mechanisms in relation to the ability to repair and survive interstrand DNA cross-links. IMPORTANCE Psoralen DNA interstrand cross-links are highly toxic lesions with antimicrobial and anticancer properties. Despite the lack of effective mechanisms for repair, cells can become resistant to cross-linking agents through mechanisms that remain poorly defined. We derived resistant mutants and identified that two gain-of-function mutations in AcrR and the alpha subunit of RNA polymerase confer high levels of resistance to E. coli treated with psoralen-UVA. Resistance conferred by AcrR mutations occurs through regulation of the AcrAB-TolC efflux pump, has an additive effect with RNA polymerase mutations, acts by reducing the formation of cross-links in vivo, and reveals a novel mechanism by which these environmentally and clinically important agents are processed by the cell.

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.

Recombination Mediator Proteins: Misnomers That Are Key to Understanding the Genomic Instabilities in Cancer.

Recombination mediator proteins have come into focus as promising targets for cancer therapy, with synthetic lethal approaches now clinically validated by the efficacy of PARP inhibitors in treating BRCA2 cancers and RECQ inhibitors in treating cancers with microsatellite instabilities. Thus, understanding the cellular role of recombination mediators is critically important, both to improve current therapies and develop new ones that target these pathways. Our mechanistic understanding of BRCA2 and RECQ began in Escherichia coli. Here, we review the cellular roles of RecF and RecQ, often considered functional homologs of these proteins in bacteria. Although these proteins were originally isolated as genes that were required during replication in sexual cell cycles that produce recombinant products, we now know that their function is similarly required during replication in asexual or mitotic-like cell cycles, where recombination is detrimental and generally not observed. Cells mutated in these gene products are unable to protect and process replication forks blocked at DNA damage, resulting in high rates of cell lethality and recombination events that compromise genome integrity during replication.

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

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.

UV-induced DNA damage disrupts the coordination between replication initiation, elongation and completion.

Replication initiation, elongation and completion are tightly coordinated to ensure that all sequences replicate precisely once each generation. UV-induced DNA damage disrupts replication and delays elongation, which may compromise this coordination leading to genome instability and cell death. Here, we profiled the Escherichia coli genome as it recovers from UV irradiation to determine how these replicational processes respond. We show that oriC initiations continue to occur, leading to copy number enrichments in this region. At late times, the combination of new oriC initiations and delayed elongating forks converging in the terminus appear to stress or impair the completion reaction, leading to a transient over-replication in this region of the chromosome. In mutants impaired for restoring elongation, including recA, recF and uvrA, the genome degrades or remains static, suggesting that cell death occurs early after replication is disrupted, leaving partially duplicated genomes. In mutants impaired for completing replication, including recBC, sbcCD xonA and recG, the recovery of elongation and initiation leads to a bottleneck, where the nonterminus region of the genome is amplified and accumulates, indicating that a delayed cell death occurs in these mutants, likely resulting from mis-segregation of unbalanced or unresolved chromosomes when cells divide.

Manganese Is Required for the Rapid Recovery of DNA Synthesis following Oxidative Challenge in Escherichia coli.

Divalent metals such as iron and manganese play an important role in the cellular response to oxidative challenges and are required as cofactors by many enzymes. However, how these metals affect replication after oxidative challenge is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. We show that the manganese-dependent recovery of DNA synthesis occurs independent of lesion repair, modestly improves cell survival, and is associated with elevated rates of mutagenesis. The Mn-dependent mutagenesis involves both replicative and translesion polymerases and requires prior disruption by H2O2 to occur. Taking these findings together, we propose that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. The data suggest that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.IMPORTANCE Iron and manganese play important roles in how cell's cope with oxygen stress. However, how these metals affect the ability of cells to replicate after oxidative challenges is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. The manganese-dependent recovery of DNA synthesis occurs independently of lesion repair and modestly improves survival, but it also increases the mutation rate in cells. The results imply that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. We propose that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.

RecBCD, SbcCD and ExoI process a substrate created by convergent replisomes to complete DNA replication.

The accurate completion of DNA replication on the chromosome requires RecBCD and structure specific SbcCD and ExoI nucleases. However, the substrates and mechanism by which this reaction occurs remains unknown. Here we show that these completion enzymes operate on plasmid substrates containing two replisomes, but are not required for plasmids containing one replisome. Completion on the two-replisome plasmids requires RecBCD, but does not require RecA and no broken intermediates accumulate in its absence, indicating that the completion reaction occurs normally in the absence of any double-strand breaks. Further, similar to the chromosome, we show that when the normal completion reaction is prevented, an aberrant RecA-mediated recombination process leads to amplifications that drive most of the instabilities associated with the two-replisome substrates. The observations imply that the substrate SbcCD, ExoI and RecBCD act upon in vivo is created specifically by two convergent replisomes, and demonstrate that the function of RecBCD in completing replication is independent of double-strand break repair, and likely promotes joining of the strands of the convergent replication forks.

Limited Capacity or Involvement of Excision Repair, Double-Strand Breaks, or Translesion Synthesis for Psoralen Cross-Link Repair in Escherichia coli.

DNA interstrand cross-links are complex lesions that covalently bind complementary strands of DNA and whose mechanism of repair remains poorly understood. In Escherichia coli, several gene products have been proposed to be involved in cross-link repair based on the hypersensitivity of mutants to cross-linking agents. However, cross-linking agents induce several forms of DNA damage, making it challenging to attribute mutant hypersensitivity specifically to interstrand cross-links. To address this, we compared the survival of UVA-irradiated repair mutants in the presence of 8-methoxypsoralen-which forms interstrand cross-links and monoadducts-to that of angelicin-a congener forming only monoadducts. We show that incision by nucleotide excision repair is not required for resistance to interstrand cross-links. In addition, neither RecN nor DNA polymerases II, IV, or V is required for interstrand cross-link survival, arguing against models that involve critical roles for double-strand break repair or translesion synthesis in the repair process. Finally, estimates based on Southern analysis of DNA fragments in alkali agarose gels indicate that lethality occurs in wild-type cells at doses producing as few as one to two interstrand cross-links per genome. These observations suggest that E. coli may lack an efficient repair mechanism for this form of damage.

Replication Rapidly Recovers and Continues in the Presence of Hydroxyurea in Escherichia coli.

In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in Escherichia coli is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases NrdEF and NrdDG or the translesion DNA polymerases II (Pol II), Pol IV, and Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, they do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was observed only under conditions where the stability of hydroxyurea was compromised and by-products known to damage DNA directly were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesis in vivo and that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.IMPORTANCE Hydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in Escherichia coli before replication rapidly recovers and continues in the presence of the drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further, we show that hydroxyurea toxicity is observed only in the presence of toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA, and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.

SbcC-SbcD and ExoI process convergent forks to complete chromosome replication.

SbcC-SbcD are the bacterial orthologs of Mre11-Rad50, a nuclease complex essential for genome stability, normal development, and viability in mammals. In vitro, these enzymes degrade long DNA palindromic structures. When inactivated along with ExoI in Escherichia coli, or Sae2 in eukaryotes, palindromic amplifications arise and propagate in cells. However, long DNA palindromes are not normally found in bacterial or human genomes, leaving the cellular substrates and function of these enzymes unknown. Here, we show that during the completion of DNA replication, convergent replication forks form a palindrome-like structural intermediate that requires nucleolytic processing by SbcC-SbcD and ExoI before chromosome replication can be completed. Inactivation of these nucleases prevents completion from occurring, and under these conditions, cells maintain viability by shunting the reaction through an aberrant recombinational pathway that leads to amplifications and instability in this region. The results identify replication completion as an event critical to maintain genome integrity and cell viability, demonstrate SbcC-SbcD-ExoI acts before RecBCD and is required to initiate the completion reaction, and reveal how defects in completion result in genomic instability.

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.

Completion of DNA replication in Escherichia coli.

The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.

Fate of the replisome following arrest by UV-induced DNA damage in Escherichia coli.

Accurate replication in the presence of DNA damage is essential to genome stability and viability in all cells. In Escherichia coli, DNA replication forks blocked by UV-induced damage undergo a partial resection and RecF-catalyzed regression before synthesis resumes. These processing events generate distinct structural intermediates on the DNA that can be visualized in vivo using 2D agarose gels. However, the fate and behavior of the stalled replisome remains a central uncharacterized question. Here, we use thermosensitive mutants to show that the replisome's polymerases uncouple and transiently dissociate from the DNA in vivo. Inactivation of α, ß, or τ subunits within the replisome is sufficient to signal and induce the RecF-mediated processing events observed following UV damage. By contrast, the helicase-primase complex (DnaB and DnaG) remains critically associated with the fork, leading to a loss of fork integrity, degradation, and aberrant intermediates when disrupted. The results reveal a dynamic replisome, capable of partial disassembly to allow access to the obstruction, while retaining subunits that maintain fork licensing and direct reassembly to the appropriate location after processing has occurred.

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.

Cellular characterization of the primosome and rep helicase in processing and restoration of replication following arrest by UV-induced DNA damage in Escherichia coli.

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion.

Mfd is required for rapid recovery of transcription following UV-induced DNA damage but not oxidative DNA damage in Escherichia coli.

Transcription-coupled repair (TCR) is a cellular process by which some forms of DNA damage are repaired more rapidly from transcribed strands of active genes than from nontranscribed strands or the overall genome. In humans, the TCR coupling factor, CSB, plays a critical role in restoring transcription following both UV-induced and oxidative DNA damage. It also contributes indirectly to the global repair of some forms of oxidative DNA damage. The Escherichia coli homolog, Mfd, is similarly required for TCR of UV-induced lesions. However, its contribution to the restoration of transcription and to global repair of oxidative damage has not been examined. Here, we report the first direct study of transcriptional recovery following UV-induced and oxidative DNA damage in E. coli. We observed that mutations in mfd or uvrA reduced the rate that transcription recovered following UV-induced damage. In contrast, no difference was detected in the rate of transcription recovery in mfd, uvrA, fpg, nth, or polB dinB umuDC mutants relative to wild-type cells following oxidative damage. mfd mutants were also fully resistant to hydrogen peroxide (H(2)O(2)) and removed oxidative lesions from the genome at rates comparable to wild-type cells. The results demonstrate that Mfd promotes the rapid recovery of gene expression following UV-induced damage in E. coli. In addition, these findings imply that Mfd may be functionally distinct from its human CSB homolog in that it does not detectably contribute to the recovery of gene expression or global repair following oxidative damage.

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.

ATP binding, ATP hydrolysis, and protein dimerization are required for RecF to catalyze an early step in the processing and recovery of replication forks disrupted by DNA damage.

In Escherichia coli, the recovery of replication following disruption by UV-induced DNA damage requires the RecF protein and occurs through a process that involves stabilization of replication fork DNA, resection of nascent DNA to allow the offending lesion to be repaired, and reestablishment of a productive replisome on the DNA. RecF forms a homodimer and contains an ATP binding cassette ATPase domain that is conserved among eukaryotic SMC (structural maintenance of chromosome) proteins, including cohesin, condensin, and Rad50. Here, we investigated the functions of RecF dimerization, ATP binding, and ATP hydrolysis in the progressive steps involved in recovering DNA synthesis following disruption by DNA damage. RecF point mutations with altered biochemical properties were constructed in the chromosome. We observed that protein dimerization, ATP binding, and ATP hydrolysis were essential for maintaining and processing the arrested replication fork, as well as for restoring DNA synthesis. In contrast, stabilization of the RecF protein dimer partially protected the DNA at the arrested fork from degradation, although overall processing and recovery remained severely impaired.

Nucleotide excision repair is a predominant mechanism for processing nitrofurazone-induced DNA damage in Escherichia coli.

Nitrofurazone is reduced by cellular nitroreductases to form N(2)-deoxyguanine (N(2)-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other N(2)-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in Escherichia coli cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 microM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.