Abstract
Invivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events invivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events athigh resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and downstream of a site-specific DNA lesion. Pol V-mediated untargeted mutations are thus spread over several hundred nucleotides, strongly eliciting genetic instability on either side of a given lesion. Consequently, post-replicative gap repair may be a source of untargeted mutations critical for gene diversification in adaptation and evolution.
Highlights
DNA damage has pervasive and deleterious effects, and cells have evolved efficient defense mechanisms to cope with DNA damage
Post-replicative gaps are repaired either by homologous recombination using the sister chromatid as a template or by translesion synthesis (TLS), a process that involves the recruitment of specialized DNA polymerases able to bypass DNA lesions (Fuchs, 2016)
By exploiting the large difference in fidelity between Pol III and Pol V, it should be possible to visualize the patches of DNA synthesized by Pol V in vivo by tracking replication errors
Summary
DNA damage has pervasive and deleterious effects, and cells have evolved efficient defense mechanisms to cope with DNA damage. Despite efficient repair, DNA polymerases occasionally encounter unrepaired DNA lesions, causing transient replication blockage. Post-replicative gaps are repaired either by homologous recombination using the sister chromatid as a template or by translesion synthesis (TLS), a process that involves the recruitment of specialized DNA polymerases able to bypass DNA lesions (Fuchs, 2016). The process of TLS is widely found from prokaryotes to eukaryotes (Goodman, 2002) This pathway is relevant to human health, because a cancer-prone human disease, xeroderma pigmentosum variant (XPV), is caused by mutations in a specialized DNA polymerase gene (Johnson et al, 1999; Masutani et al, 1999). In contrast to typical repair pathways that accurately repair damage, TLS is intrinsically mutation prone because of the low replication fidelity of the specialized polymerases. To avoid unnecessary mutagenic risk, TLS polymerases are tightly controlled at transcriptional and/or post-translational levels (Goodman, 2002)
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