| Ultraviolet irradiation induces DNA damage that can block the progression of the replication machinery or generate gaps in the nascent DNA, depending on which template the lesion is found. Characterization of the mechanisms by which DNA synthesis resumes and the integrity of the template restored following UV-induced damage is important to understand how genome stability is maintained in all organisms. When progression of replication is blocked at a UV-induced lesion, several lines of evidence suggest that the resumption of replication requires that the blocking lesion is repaired. A remaining question is how or if the nascent DNA and replication machinery are displaced so repair enzymes can gain access to the damaged DNA and effect repair. Biochemical studies have shown that RecG and RuvABC can catalyze this reaction on synthetic DNA substrates and it has been proposed that one or both may be required to catalyze this reaction in vivo. In this study, it is shown that ruvAB, ruvC, and recG mutants resume DNA synthesis at a time and rate that is similar to wild-type and that neither gene product is required to maintain the structural integrity of blocked replication forks, indicating that RuvABC and RecG are not essential for the resumption of replication. Although DNA synthesis continued, ruvAB and ruvC mutants accumulated unresolved Holliday junctions during replication after UV irradiation, leading to a general deterioration in the integrity of the genomic DNA, suggesting a potential role for RuvABC in processing Holliday junctions that accumulate following replication past lesions that do not arrest the replication machinery. Structural intermediates induced by UV-irradiation were also characterized by two-dimensional agarose gel electrophoresis in recG, recJ, recQ, xonA, recBC, and recD mutants. It is shown that recJ, recQ, and xonA mutants form UV-induced intermediates that resemble those observed in wild-type cells, but persist for a modestly longer period prior to their resolution. recBC mutants form UV-induced intermediates that appear similar to wild-type, but these intermediates persist throughout the times that were examined. In contrast, recD mutants form unique intermediates both in the presence and absence of UV irradiation that are not observed in wild-type. |
