Rad18-mediated PCNA ubiquitination has been proposed to promote Polζ- and Rev1-dependent DNA synthesis. PCNA ubiquitination has been shown to mediate Rev1-PCNA interaction [41
]. However, localization of Polζ and Rev1 to DSB lesions does not require Rad18 function or PCNA ubiquitination [8
]. It has thus remained possible that Polζ and Rev1 contribute to DSB repair independently of PCNA ubiquitination. In this study, we have used a system in which cells receive pure DSBs after HO
expression and found that the Rad18-mediated PCNA ubiquitination activates the Polζ/Rev1 pathway in DSB repair as well. These observations support the model in which Polζ-Rev1 first accumulates at regions near DSBs and then interacts with ubiquitinated PCNA to facilitate DNA synthesis. Exposure to DSB inducing agents, for example ionization radiation (IR), also induces single-strand breaks, base modifications and DNA adducts, which can be repaired by the TLS pathway. For example, the introduction of a rev1Δ
mutation significantly reduces viability of rad52Δ hdf1Δ
double mutant cells after IR (data not shown). Therefore, it is difficult to characterize functions of Polζ-Rev1 and Rad18 in DSB repair using IR. HO endonuclease, which generates pure DSBs, enabled us to investigate a role of Polζ-Rev1 and Rad18 in DSB repair.
If simple re-joining of DNA ends does not occur, DSBs are processed by exonuclease activities, generating ssDNA tract at the DSB ends. The Rad6-Rad18 complex has been shown to bind to single-stranded DNA in vitro
]. This biochemical property of the Rad6-Rad18 complex is consistent with our observation that Rad18 localizes to DSBs. During the revision of this paper, Davies et al. [42
] also reported that Rad18 localizes to DSBs. Requirement of Rad18 for DSB repair has been shown in chicken DT 40 cells [43
], suggesting that Rad18 associates with DSB lesions in higher eukaryotes as well. The Rad6-Rad18 complex has been proposed to ubiquitinate PCNA loaded on DNA [19
], because only a small fraction of PCNA is modified after DNA damage. Even though Rad18 localizes to DSBs, PCNA modification has not been detected in cells suffering HO-induced or Eco
RI-induced DSBs by immunoblotting analysis (data not shown)[44
]. It is not known what fraction of PCNA is loaded on DNA at a DSB lesion or how many DSBs are generated after Eco
RI expression. If only a few PCNA molecules are loaded on DNA, it might be difficult to detect PCNA ubiquitination.
Annealing of ssDNA tracts at DNA ends may generate gaps with 3’-termini. Replication factor C can load PCNA on gapped DNA substrates [45
]. The Rad6-Rad18 and Polζ-Rev1 complexes accumulate at sites near DSB ends [8
]. Once loaded, PCNA could be ubiquitinated by the Rad6-Rad18 complex. In turn, ubiquitinated PCNA might stimulate the activity of nearby Polζ or Rev1 [19
]. Previous studies have demonstrated that Pol4 plays an important role in gap-filling, by extending the DNA strand from mismatched 3’-termini [39
]. Similarly, Polζ efficiently extends from mismatched termini [46
]. Polζ is a processive enzyme [46
], whereas Pol4 is not [40
]. Thus, Polζ-Rev1 and Pol4 could play distinct roles in gap-filling during NHEJ. Recent studies have established the model in which the cyclin-dependent kinase, Cdc28, stimulates resection of DNA ends to generate ssDNA tracts at DSBs [47
]. This model explains why NHEJ operates preferentially in G1. In the absence of DNA degradation, however, NHEJ could rejoin the DNA ends without gap-filling. Thus, DNA polymerase activities might be more dispensable for NHEJ in G1 phase than in other cell-cycle stages. Consistently, Rev1 and Rad18 do not play an apparent role in repair of HO-induced DSBs in G1 (data not shown), although Ku plays a critical role [49
]. Since Mec1 localizes to sites of DNA damage by interacting with RPA-coated ssDNA [50
], Mec1 associate with DSBs in S and G2/M phase more efficiently than in G1 phase [52
]. We have shown that Mec1-dependent phosphorylation promotes Polζ-Rev1 association with DSBs [8
]. Mec1-mediated Polζ-Rev1 accumulation might assure efficient gap-filling during NHEJ in S or G2/M phase.
PCNA ubiquitination activates the Polη pathway as well as the Polζ/Rev1 pathway [16
]. However, Polη (Rad30) is dispensable for our DSB repair assay. These findings suggest that PCNA ubiquitination does not always activate the Polη pathway. Association of Polη with the HO-induced DSB has not been detected by ChIP assay (data not shown), whereas Polζ and Rev1 association is observed [8
]. Association of human Polη with damage sites is dependent on PCNA ubiquitination [21
]. As discussed above, ubiquitination of PCNA has not been detected after DSB induction. It is therefore possible that each DSB lesion contains only a few ubiquitinated PCNA molecules. In this case, Polη might fail to accumulate at DSBs in budding yeast. By contrast, Polζ-Rev1 accumulates at DSBs independently of PCNA ubiquitination. Therefore, Polζ-Rev1 could interact with ubiquitinated PCNA at DSBs.
Although there are only two TLS polymerases (Polζ and Polη) in budding yeast, human cells express more varied TLS polymerases including Polκ and Polι [1
]. These TLS polymerases may play specific roles at sites of DNA damage, because they possess different biochemical properties. However, it still remains unclear how these TLS polymerases act coordinately in DNA repair. Our results suggest that ubiquitination-independent accumulation helps Polζ-Rev1 to act differently from Polη in budding yeast.