Rad18 Regulates DNA Polymerase κ and Is Required for Recovery from S-Phase Checkpoint-Mediated Arrest

Abstract

ABSTRACT We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polκ to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polκ in cells treated with the bulky adduct-forming genotoxin benzo[ a ]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polκ. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polκ. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polκ in a DNA damage-independent manner. Therefore, association of Polκ with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18 −/− transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18 −/− cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18 -deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polκ. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polκ to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.