Eukaryotic stress induced mutagenesis is limited by a local control of Translesion Synthesis

Abstract

The DNA Damage Response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this report, we raise the question whether TLS is controlled locally, or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that: i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, ii) a single lesion is enough to induce PCNA ubiquitination, and iii) PCNA ubiquitination is an absolute requirement for TLS to occur. More importantly, we show that global PCNA ubiquitination that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.

Opposite roles of Rad5 in DNA damage tolerance: playing in both error-free and mutagenic lesion bypass

“…The same increase was observed for the mutation affecting only the ubiquitin ligase activity ( RAD5IA ubi ), indicating that it is the lack of PCNA poly-ubiquitination in the two tested mutants that is responsible for the increase in TLS. This increase is similar to the one previously observed in the absence of ubc13 [18,19]. We have previously described a competition between TLS and DA: in the absence of polyubiquitination of PCNA, DA is inhibited favoring TLS.…”

“…• Rad5 interacHon with Rev1 is required for Polζ-TLS For both (6-4)TT photoproduct and N2dG-AAF lesions, the RAD5 allele unable to bind Rev1 (RAD5FN Rev1 ) causes a very strong decrease in the level of TLS through those lesions (Figure 2A and B). As we have shown previously [18,19], TLS bypass of those lesions relies Untreated 4NQO -0.015µM 4NQO -0.03µM almost exclusively on the TLS polymerases Rev1 and Pol ζ. This indicates that the interacPon of Rev1 with Rad5 is criPcal for its TLS acPvity in vivo.…”

“…We observed a slight decrease in TLS for the N2dG-AAF lesions, and a very moderate decrease for the the TT(6-4) photoproduct compared to the parental strain. It is important to note that in the rad5 Δ strain, no polyubiquitination of PCNA occurs: we could therefore expect in these strains a strong increase of TLS as observed in the RAD5DE ubi-helic or RAD5IA ubi mutants or in the ubc13 Δ mutant [18,19]. However, due to the absence of Rad5 and its function of recruiting Rev1, TLS does not increase in this strain.…”