Role of budding yeast Rad18 in repair of HO-induced double-strand breaks.
The Rad6-Rad18 complex mono-ubiquitinates proliferating cell nuclear antigen (PCNA) at the lysine 164 residue after DNA damage and promotes DNA polymerase eta (Poleta)- and Polzeta/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 Polzeta and Rev1 localize to HO-induced DSBs in a Mec1-dependent manner and promote Ku-dependent DSB repair. However, Polzeta and Rev1 localize to DSBs independently of PCNA ubiquitination. Here we provide evidence indicating that Rad18-mediated PCNA ubiquitination stimulates DNA synthesis by Polzeta and Rev1 in repair of HO-induced DSBs. Ubiquitination defective PCNA mutation or rad18Delta mutation confers the same DSB repair defect as rev1Delta mutation. Consistent with a role in DSB repair, Rad18 localizes to HO-induced DSBs in a Rad6-dependent manner. Unlike Polzeta or Rev1, Poleta is dispensable for repair of HO-induced DSBs. Ku and DNA ligase IV constitute a central NHEJ pathway. We also show that Polzeta and Rev1 act in the same pathway as DNA ligase IV, suggesting that Polzeta and Rev1 are involved in DNA synthesis during NHEJ. Our results suggest that Polzeta-Rev1 accumulates at regions near DSBs independently of PCNA ubiquitination and then interacts with ubiquitinated PCNA to facilitate DNA synthesis.
Hirano
Yukinori
Y
Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, United States.
Reddy
Jayant
J
Sugimoto
Katsunori
K
D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C000607891:DNL4 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D011088:DNA Ligases; D000072481:DNA Ligase ATP