Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer

Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichiacoli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.     

RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli

After UV irradiation, recA mutants fail to recover replication, and a dramatic and nearly complete degradation of the genomic DNA occurs. Although the RecBCD helicase/nuclease complex is known to mediate this catastrophic DNA degradation, it is not known how or where this degradation is initiated. Previous studies have speculated that RecBCD targets and initiates degradation from the nascent DNA at replication forks arrested by DNA damage. To test this question, we examined which enzymes were responsible for the degradation of genomic DNA and the nascent DNA in UV-irradiated recA cells. We show here that, although RecBCD degrades the genomic DNA after UV irradiation, it does not target the nascent DNA at arrested replication forks. Instead, we observed that the nascent DNA at arrested replication forks in recA cultures is degraded by RecJ/RecQ, similar to what occurs in wild-type cultures. These findings indicate that the genomic DNA degradation and nascent DNA degradation in UV-irradiated recA mutants are mediated separately through RecBCD and RecJ/RecQ, respectively. In addition, they demonstrate that RecBCD initiates degradation at a site(s) other than the arrested replication fork directly.     

Action of RecBCD enzyme on Holliday structures made by RecA

In vitro, Escherichia coli RecA protein acts upon gapped and partially homologous linear duplex DNA to generate recombination products linked by Holliday junctions. When strand exchange reactions are supplemented with purified RecBCD enzyme, we observe the formation of products that resemble "patch" recombinants. The formation of "splice" recombinant products was not observed. The individual subunits, RecB, RecC, or RecD, had no effect on RecA protein-mediated strand exchange nor on the Holliday junctions formed in the reaction. Analysis of the way in which patch products arise indicates exonucleolytic digestion of the linear arms of the recombination intermediates (alpha-structures) by RecBCD enzyme. We find no evidence for specific resolution events at the site of the Holliday junction by RecBCD enzyme using these DNA substrates.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

Chromosomal lesion suppression and removal in Escherichia coli via linear DNA degradation

RecBCD is a DNA helicase/exonuclease implicated in degradation of foreign linear DNA and in RecA-dependent recombinational repair of chromosomal lesions in E. coli. The low viability of recA recBC mutants vs. recA mutants indicates the existence of RecA-independent roles for RecBCD. To distinguish among possible RecA-independent roles of the RecBCD enzyme in replication, repair, and DNA degradation, we introduced wild-type and mutant combinations of the recBCD chromosomal region on a low-copy-number plasmid into a DeltarecA DeltarecBCD mutant and determined the viability of resulting strains. Our results argue against ideas that RecBCD is a structural element in the replication factory or is involved in RecA-independent repair of chromosomal lesions. We found that RecBCD-catalyzed DNA degradation is the only activity important for the recA-independent viability, suggesting that degradation of linear tails of sigma-replicating chromosomes could be one of the RecBCD's roles. However, since the weaker DNA degradation capacity due a combination of the RecBC helicase and ssDNA-specific exonucleases restores viability of the DeltarecA DeltarecBCD mutant to a significant extent, we favor suppression of chromosomal lesions via linear DNA degradation at reversed replication forks as the major RecA-independent role of the RecBCD enzyme.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

RecG helicase promotes DNA double-strand break repair

Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood. To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA.     

Chromosome demise in the wake of ligase-deficient replication

Bacterial DNA ligases, NAD⁺‐dependent enzymes, are distinct from eukaryotic ATP‐dependent ligases, representing promising targets for broad‐spectrum antimicrobials. Yet, the chromosomal consequences of ligase‐deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase‐deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double‐strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non‐allelic double‐strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double‐strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase‐deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double‐strand breaks and then into irreparable double‐strand gaps may be behind lethality of any DNA damaging treatment.     

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.     

Replication arrest is a major threat to growth at low temperature in Antarctic Pseudomonas syringae Lz4W

Chromosomal damage was detected previously in the recBCD mutants of the Antarctic bacterium Pseudomonas syringae Lz4W, which accumulated linear chromosomal DNA leading to cell death and growth inhibition at 4°C. RecBCD protein generally repairs DNA double‐strand breaks by RecA‐dependent homologous recombination pathway. Here we show that ΔrecA mutant of P. syringae is not cold‐sensitive. Significantly, inactivation of additional DNA repair genes ruvAB rescued the cold‐sensitive phenotype of ΔrecBCD mutant. The ΔrecA and ΔruvAB mutants were UV‐sensitive as expected. We propose that, at low temperature DNA replication encounters barriers leading to frequent replication fork (RF) arrest and fork reversal. RuvAB binds to the reversed RFs (RRFs) having Holliday junction‐like structures and resolves them upon association with RuvC nuclease to cause linearization of the chromosome, a threat to cell survival. RecBCD prevents this by degrading the RRFs, and facilitates replication re‐initiation. This model is consistent with our observation that low temperature‐induced DNA lesions do not evoke SOS response in P. syringae. Additional studies show that two other repair genes, radA (encoding a RecA paralogue) and recF are not involved in providing cold resistance to the Antarctic bacterium.     

A gene that regulates DNA replication in response to DNA damage is located on human chromosome 4q.

Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.     

Replisome-mediated Translesion Synthesis and Leading Strand Template Lesion Skipping Are Competing Bypass Mechanisms

A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3′ → 5′ proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its “little finger” domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions.     

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor,Is a Msh2-Msh3-stimulated Endonuclease

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.     

Identification of novel initiation sites for human DNA replication around ARSH1, a previously characterized yeast replicator

Replication of mammalian chromosomes depends on the activation of a large number of origins of DNA replication distributed along the chromosomes. We have focused our attention on a human DNA region, named ARSH1, localized to chromosome 2, that had been previously shown to act as an episomal origin in the yeast Saccharomyces cerevisiae. In the present study we have used a nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 5 kb region encompassing ARSH1. This analysis applied to a 1-1.4 kb nascent DNA strand fraction isolated from normal skin fibroblasts revealed the presence of two major initiations sites surrounding the ARSH1 region. With an equivalent DNA fraction obtained from HeLa cells, in addition to these sites, a broad initiation profile was observed which included the ARSH1 region. This DNA region however was not sufficient to support episomal replication of an ARSH1-containing plasmid transfected into HeLa cells.     

RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli

RuvABC is a complex that promotes branch migration and resolution of Holliday junctions. Although ruv mutants are hypersensitive to UV irradiation, the molecular event(s) that necessitate RuvABC processing in vivo are not known. Here, we used a combination of two-dimensional gel analysis and electron microscopy to reveal that although ruvAB and ruvC mutants are able to resume replication following arrest at UV-induced lesions, molecules that replicate in the presence of DNA damage accumulate unresolved Holliday junctions. The failure to resolve the Holliday junctions on the fully replicated molecules correlates with a delayed loss of genomic integrity that is likely to account for the loss of viability in these cells. The strand exchange intermediates that accumulate in ruv mutants are distinct from those observed at arrested replication forks and are not subject to resolution by RecG. These results indicate that the Holliday junctions observed in ruv mutants are intermediates of a repair pathway that is distinct from that of the recovery of arrested replication forks. A model is proposed in which RuvABC is required to resolve junctions that arise during the repair of a subset of nonarresting lesions after replication has passed through the template.     

Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA.

We have carried out a series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli. In many respects, these effects differ greatly from those previously described for non-density-labeled (light) phage. Moreover, our results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication, strand displacement, and a considerable amount of DNA replication. UV irradiation of 5-bromodeoxyuridine-labeled phage results in single-stranded breaks in a linear, dose-dependent manner (1.3 to 1.5 breaks per genomic strand per lethal hit). This damage does not interfere with injection of the phage genome, but some of the UV-irradiated heavy phage DNA undergoes additional intracellular breakdown (also dose dependent). However, a minority (25%) of the injected parental DNA is protected, maintaining its preinjection size. This protected moiety is associated with a replicative complex of DNA and proteins, and is more efficiently replicated than is the parental DNA not so associated. Most of the progeny DNA is also found with the replicative complex. The 5-bromodeoxyuridine of heavy phage DNA is debrominated by UV irradiation, resulting in uracil which is removed by host uracil glycosylase. Unlike the simple gap-filling repair synthesis after infection with UV-irradiated light phage, the repair replication of UV-irradiated heavy phage is extensive as determined by density shift of the parental label in CsC1 gradients. The newly synthesized segments are covalently attached to the parental fragments. The repair replication takes place even in the presence of chloramphenicol, a protein synthesis inhibitor, suggesting it is host mediated. Furthermore, the extent of the repair replication is greater at higher doses of UV irradiation applied to the heavy phage. This abundant synthesis results ultimately in dispersion of the parental sequences as short stretches in the midst of long segments of newly synthesized progeny DNA. Together, the extensive replication and the resulting distribution pattern of parental sequences, without significant solubilization of parental label, are most consistent with a model of repair synthesis in which the leading strand displaces, rather than ligates to, the encountered 5' end.     

Analysis of the Activities of RAD54, a SWI2/SNF2 Protein,Using a Specific Small-molecule Inhibitor

RAD54, an important homologous recombination protein, is a member of the SWI2/SNF2 family of ATPase-dependent DNA translocases. In vitro, RAD54 stimulates RAD51-mediated DNA strand exchange and promotes branch migration of Holliday junctions. It is thought that an ATPase-dependent DNA translocation is required for both of these RAD54 activities. Here we identified, by high-throughput screening, a specific RAD54 inhibitor, streptonigrin (SN), and used it to investigate the mechanisms of RAD54 activities. We found that SN specifically targets the RAD54 ATPase, but not DNA binding, through direct interaction with RAD54 and generation of reactive oxygen species. Consistent with the dependence of branch migration (BM) on the ATPase-dependent DNA translocation of RAD54, SN inhibited RAD54 BM. Surprisingly, the ability of RAD54 to stimulate RAD51 DNA strand exchange was not significantly affected by SN, indicating a relatively smaller role of RAD54 DNA translocation in this process. Thus, the use of SN enabled us to identify important differences in the effect of the RAD54 ATPase and DNA translocation on two major activities of RAD54, BM of Holliday junctions and stimulation of DNA pairing.     

Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome

The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.