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...

Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by twodimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including recF, recO, recR, recJ, recG, and xonA. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.All cells must accurately duplicate their genomes in order to reproduce. However, even under normal conditions, a variety of biologically important impediments, such as base alterations, DNA adducts, DNA strand breaks, DNA-bound proteins, secondary structures in the DNA, or even limitations in the processivity of the replication machinery itself, may impair the ability of the replication machinery to complete its task (for reviews, see references 14 and 16). Each of these impediments poses unique challenges for the cell and may stall, block, or disrupt the replication machinery. Although the specific structure and nature of how the replication holoenzyme arrests in each of these situations are not known, it is reasonable to assume that the mechanisms by which replication recovers may vary, depending on the nature of the obstacle. In order to understand how genomic stability is maintained throughout the life span of an organism, it is important to characterize how arrested replication forks are accurately processed and resume in each of these situations.UV-induced DNA damage has frequently been used as a model to address the general question of how replication recovers when it is blocked by DNA damage, and this damage has been extensively characterized. Irradiation with 254-nm light induces DNA lesions that block the progression of the replication machinery (6,15,48). In Escherichia coli, RecA and several of the RecF pathway gene products are required to maintain and...

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