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...

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...