Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint-mediated arrest.
We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polkappa to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polkappa 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 Polkappa. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polkappa. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polkappa in a DNA damage-independent manner. Therefore, association of Polkappa 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 Polkappa. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polkappa to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.
Bi
Xiaohui
X
Department of Genetics and Genomics, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, USA.
Barkley
Laura R
LR
Slater
Damien M
DM
Tateishi
Satoshi
S
Yamaizumi
Masaru
M
Ohmori
Haruo
H
Vaziri
Cyrus
C
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002273:Carcinogens / Q000633:toxicity; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016196:S Phase / Q000187:drug effects / Q000235:genetics; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D000099270:Y-Family DNA Polymerases
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D064007:Ataxia Telangiectasia Mutated Proteins; D002273:Carcinogens; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D049452:Green Fluorescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C576597:ATR protein, human; C000605867:CHEK1 protein, human; C000605869:Chek1 protein, mouse; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases