Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli.

Transformation of Escherichia coli with purified plasmids containing DNA damage is frequently used as a tool to characterize repair pathways that operate on chromosomes. In this study, we used an assay that allowed us to quantify plasmid survival and to compare how efficiently various repair pathways operate on plasmid DNA introduced into cells relative to their efficiency on chromosomal DNA. We observed distinct differences between the mechanisms operating on the transforming plasmid DNA and the chromosome. An average of one UV-induced lesion was sufficient to inactivate ColE1-based plasmids introduced into nucleotide excision repair mutants, suggesting an essential role for repair on newly introduced plasmid DNA. By contrast, the absence of RecA, RecF, RecBC, RecG, or RuvAB had a minimal effect on the survival of the transforming plasmid DNA containing UV-induced damage. Neither the presence of an endogenous homologous plasmid nor the induction of the SOS response enhanced the survival of transforming plasmids. Using two-dimensional agarose-gel analysis, both replication- and RecA-dependent structures that were observed on established, endogenous plasmids following UV-irradiation, failed to form on UV-irradiated plasmids introduced into E. coli. We interpret these observations to suggest that the lack of RecA-mediated survival is likely to be due to inefficient replication that occurs when plasmids are initially introduced into cells, rather than to the plasmid's size, the absence of homologous sequences, or levels of recA expression.

Requirement for uracil-DNA glycosylase during the transition to late-phase cytomegalovirus DNA replication.

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.

Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation.

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-(beta-D-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unit-length 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.