Determinants of replicative DNA-damage bypass in budding yeast
Minca, Eugen Catalin
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The faithful replication of the genetic material is challenged by unrepaired DNA damage that threatens the genomic stability and cell survival. DNA lesions can cause replication fork stalling, which is usually inferred and seldom observed directly. Fork stalling is thought to be relieved by DNA-damage-bypass mechanisms that are not well understood. Previous work showed that cell exposure to the alkylating compound adozelesin results in stalling of replication forks at a specific active origin site. We investigated the generality and the basis for fork stalling at replication origins in response to bulky lesions occupying the DNA minor groove. DNA alkylation by adozelesin and 4NQO induced site-specific fork stalling at various early-firing replication origins, but not at late origins or at inactive origins. Site-specific fork stalling was dependent on local origin activity and preceded the formation of recombination-like, X-shaped DNA structures at early replication origins. Inactivation of the intra-S-phase checkpoint alleviated but did not eliminate the DNA-damage-induced fork stalling at early origin sites, and enhanced the stalling and the X-DNA formation at an unrepressed late-firing replication origin. These results suggest that the fork stalling occurs at the first lesion encountered in the proximity of early-firing origins and activates damage-bypass mechanisms which prevent fork stalling at subsequent lesions. Multiple DNA damage repair and bypass pathways promote cell survival of genotoxic stress. DNA-damage bypass is regulated by a series of PCNA post-translational modifications which control error-prone or error-free pathways and prevent harmful DNA rearrangements. Our results show that the RAD18/RAD5 -controlled error-free DNA-damage-bypass pathway is essential for survival and replication completion in the presence of bulky lesions that occupy the DNA minor groove. Homologous recombination, but not nucleotide-excision repair, functioned in the RAD5 -dependent error-free bypass pathway for DNA damage sensitivity. Inactivation of RAD18 or other error-free bypass genes resulted in abnormal replication intermediates upon alkylation-induced fork stalling. In the absence of Rad5, elimination of post-translational modifications at PCNA Lys-164 activated an alternative damage-bypass pathway enhancing both cell survival and replication completion in the presence of DNA damage in a rad5Δ mutant. The product of the RAD5 gene, Rad5, is hypothesized to control error-free DNA-damage bypass via a replication template switch, while avoiding harmful DNA rearrangements through the Srs2 anti-recombinase. Our results show that Rad5 and recombination factors function coordinately in restarting forks stalled by DNA damage and completing chromosomal replication. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures at fork stalling sites during replication initiation. Cells lacking Rad5 or recombination factors also lacked X-DNA and were defective in restarting stalled replication forks, which then degenerated into abnormal DNA structures. In the rad5Δ mutant, the absence of Srs2, or the elimination of post-translational modifications at PCNA Lys-164, restored X-DNA structures at stalled forks and the completion of chromosomal DNA replication. Our findings support a model in which Rad5 mediates a template switch at stalled replication forks through homologous recombination between sister chromatids.