DNA lesions that block replication are a primary cause of rearrangements, mutations, and lethality in all cells. After ultraviolet (UV)-induced DNA damage in Escherichia coli, replication recovery requires RecA and several other recF pathway proteins. To characterize the mechanism by which lesion-blocked replication forks recover, we used two-dimensional agarose gel electrophoresis to show that replication-blocking DNA lesions induce a transient reversal of the replication fork in vivo. The reversed replication fork intermediate is stabilized by RecA and RecF and is degraded by the RecQ-RecJ helicase-nuclease when these proteins are absent. We propose that fork regression allows repair enzymes to gain access to the replication-blocking lesion, allowing processive replication to resume once the blocking lesion is removed.

The human cytomegalovirus UL97 kinase, an important target of antiviral therapy, has an impact on at least two distinct phases of viral replication. Compared with wild-type virus, the UL97 deletion mutant exhibits an early replication defect that reduces DNA accumulation by 4-to 6-fold, as well as a late capsid maturation defect responsible for most of the observed 100-to 1000-fold reduction in replication. Block-release experiments with the antiviral 2-bromo-5,6-dichloro-1-(␤-D-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unitlength genomes remained unaffected, progeny mutant virus maturation was delayed, with accumulation of progeny at significantly reduced levels compared with wild type after release of this block. Transmission electron microscopy confirmed the aberrant accumulation of empty A-like capsids containing neither viral DNA nor an internal scaffold structure, consistent with a failure to stably package DNA in mutant virus-infected cells. The function of UL97 in DNA synthesis as well as capsid assembly suggests that protein phosphorylation mediated by this herpesvirus-conserved kinase increases the efficiency of these two distinct phases of virus replication.

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.replication completion | double-strand break repair | RecBCD | homologous recombination | SbcDC

DNA lesions that arrest replication can lead to rearrangements, mutations, or lethality when not processed accurately. After UVinduced DNA damage in Escherichia coli, RecA and several recF pathway proteins are thought to process arrested replication forks and ensure that replication resumes accurately. Here, we show that the RecJ nuclease and RecQ helicase, which partially degrade the nascent DNA at blocked replication forks, are required for the rapid recovery of DNA synthesis and prevent the potentially mutagenic bypass of UV lesions. In the absence of RecJ, or to a lesser extent RecQ, the recovery of replication is significantly delayed, and both the recovery and cell survival become dependent on translesion synthesis by polymerase V. The RecJ-mediated processing is proposed to restore the region containing the lesion to a form that allows repair enzymes to remove the blocking lesion and DNA synthesis to resume. In the absence of nascent DNA processing, polymerase V can synthesize past the lesion to prevent lethality, although this occurs with slower kinetics and a higher frequency of mutagenesis.mutagenesis ͉ nucleotide excision repair I rradiation of cells with UV light (254 nm) induces DNA lesions that can arrest replication forks (1). 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 after UV-induced DNA damage. Although nucleotide excision repair is generally considered to be error free, the processes of translesion synthesis and recombination can be associated with mutagenesis or rearrangements, making it important to identify the order and conditions that determine when each process is employed at the arrested fork. In Escherichia coli, the robust recovery of DNA replication after UV-induced arrest largely depends on lesion removal by the nucleotide excision repair enzymes (1-4). Cells mutated in any of these gene products are unable to remove lesions from the genome and the recovery of DNA synthesis is severely impaired, resulting in elevated levels of recombination, mutagenesis, and lethality (1,(3)(4)(5).Several studies suggest that translesion synthesis by polymerase (Pol) V can also contribute to the recovery at UV-arrested forks. E. coli have three damage-inducible DNA polymerases, Pol II (polB), Pol IV (dinB), and Pol V (umuD and umuC), that have multiple homologues in both prokaryotes and eukaryotes (6). These polymerases can incorporate nucleotides opposite to specific DNA lesions with higher efficiencies than the replicative polymerase, Pol III (7-9). After UV-induced damage, Pol V, but not Pol II or IV, increases cell survival and is responsible for essentially all of the UV-induced mutagenesis that occurs after irradiation (2, 7, 10, 11). Additionally, after higher doses of UV irradiation that begin to reduce the survival of wild-type cells, Pol V contributes to the rate that DNA synthesis recovers and that nascent-strand gaps are joined, indicating that Pol...

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...

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 , 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.

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA‐like recombinases and displacing single‐stranded DNA‐binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled‐coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so‐called ‘Lobe II’ subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double‐stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP‐dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.

Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.Uracil incorporation into DNA arises through misincorporation of dUTP by DNA polymerase (2, 51, 54) or from spontaneous deamination of cytosine creating U:G base pair mismatches that resolve into A:T transition mutations upon further rounds of replication (23, 44). To avoid the potential mutagenenic impact of uracil, free-living organisms such as Escherichia coli, yeast, and human beings encode a uracil-DNA glycosylase (UNG) that excises this base from DNA (20,33,37,52). A homolog of the mammalian enzyme is encoded by all poxviruses and herpesviruses, including cytomegaloviruses (CMV), and is a highly conserved in evolution (8,25,38,39,50). CMV UL114 is the most highly conserved open reading frame in mammalian herpesviruses and retains approximately 40% identity with the major UNG expressed in human cells. Interestingly, although the major form of UNG seems to be dispensable in free-living organisms because of backup uracilexcising activities (3,6,35), viral UNG mutants are impaired in their ability to replicate efficiently under certain conditions (8,25,38,39,50). CMV UNG substitution mutant RC2620 was previously shown to replicate poorly in permissive human fibroblasts (HF cells) due to a delay in viral DNA accumulation (38). This phenotype suggested a specialized role for uracil excision during viral DNA replication.DNA replication in CMV and other herpesviruses is thought to proceed as a biphasic process (22). Origin-specific initiation on a circularized input genome leads to an early, theta mechanism that later undergoes a switch to a rolling-circle form of replication (1,16,17,22,40,48,49). Rolling-circle replication is responsible for the bulk of viral DNA produced during infection. This switch is believed to be a requisite step in replication, but little is known about the process or how it is regulated (22). Viral DNA replication is a highly recombinagenic process (7,41,55), with late replication, in particular, accompanied by the accumulation of complex branched DNA structures (43, 45). Any role for viral UNG in these processes remains poorly understood and is complicated by the fact that the viral enzyme is dispensabl...