Role of budding yeast Rad18 in repair of HO-induced double-strand breaks

Abstract

The Rad6–Rad18 complex mono-ubiquitinates proliferating cell nuclear antigen (PCNA) at the lysine 164 residue after DNA damage and promotes DNA polymerase η (Polη)- and Polζ/Rev1-dependent DNA synthesis. Double-strand breaks (DSBs) of DNA can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ), both of which require new DNA synthesis. HO endonuclease introduces DSBs into specific DNA sequences. We have shown that Polζ and Rev1 localize to HO-induced DSBs in a Mec1-dependent manner and promote Ku-dependent DSB repair. However, Polζ and Rev1 localize to DSBs independently of PCNA ubiquitination. Here we provide evidence indicating that Rad18-mediated PCNA ubiquitination stimulates DNA synthesis by Polζ and Rev1 in repair of HO-induced DSBs. Ubiquitination defective PCNA mutation or rad18Δ mutation confers the same DSB repair defect as rev1Δ mutation. Consistent with a role in DSB repair, Rad18 localizes to HO-induced DSBs in a Rad6-dependent manner. Unlike Polζ or Rev1, Polη is dispensable for repair of HO-induced DSBs. Ku and DNA ligase IV constitute a central NHEJ pathway. We also show that Polζ and Rev1 act in the same pathway as DNA ligase IV, suggesting that Polζ and Rev1 are involved in DNA synthesis during NHEJ. Our results suggest that Polζ–Rev1 accumulates at regions near DSBs independently of PCNA ubiquitination and then interacts with ubiquitinated PCNA to facilitate DNA synthesis.