Abstract
During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances, the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA-damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here, we show that in addition to translesion synthesis and strand-transfer DDT mechanisms, an alternative repair mechanism ("salvage recombination") that copies information from the sister chromatid is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1.IMPORTANCE PCNA, the ring that encircles DNA maintaining the processivity of DNA polymerases, is modified by ubiquitin and SUMO. Whereas ubiquitin is required for bypassing lesions through the DNA damage tolerance (DDT) pathways, we show here that SUMOylation represses another pathway, salvage recombination. The Srs2 helicase is recruited to SUMOylated PCNA and prevents the salvage pathway from acting. The pathway can be induced by overexpressing the PCNA unloader Elg1, or the homologous recombination protein Rad52. Our results underscore the role of PCNA modifications in controlling the various bypass and DNA repair mechanisms.
Highlights
During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions
Evidence for a Rad18- and Rad5-independent recombinational repair mechanism, which uses information from the sister chromatid to allow repair of damage sites, has been documented and was termed the salvage pathway [15, 16, 51, 52]. This mechanism requires Rad51 and Rad52 to invade the sister chromatid DNA and Sgs1 to resolve the joint molecules crated by the template switch [14]
The role for Srs2 in regulating the salvage pathway has been deduced from studies showing that deletion of SRS2, to ⌬siz1, can suppress the DNA damage sensitivity of DNA damage tolerance (DDT) mutants
Summary
During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. The first is activated by monoubiquitylation at lysine 164 of PCNA and is mediated by the E2-conjugating enzyme Rad and the E3-ubiquitin ligase Rad18 This ubiquitylation takes place at sites of fork arrest where replication protein A (RPA) accumulates [5, 6] and promotes the exchange of the replicative DNA polymerase by a translesion DNA polymerase able to bypass the DNA lesion by synthesizing DNA in an error-prone manner in most of the cases [7,8,9,10] (translesion synthesis [TLS]). This pathway is considered a last resort, as unchecked homologous recombination may generate genome instability [30] They are sometimes presented as three clear subpathways, the relationship between TLS, TS, and SR is still mysterious, and proteins may be involved in more than one category: for example, Rad, which is required to initiate the TS subpathway by polyubiquitylating PCNA, plays a role in the recruitment of TLS polymerases [31]. The SR pathway requires Rad, Rad, Rad, Sgs, and Elg activities
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