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Molecular and Cellular Biology, May 2006, p. 3527-3540, Vol. 26, No. 9
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.9.3527-3540.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Xiaohui Bi,^1, Laura R. Barkley,^1, Damien M. Slater,¹ Satoshi Tateishi,² Masaru Yamaizumi,² Haruo Ohmori,³ and Cyrus Vaziri^1*

Department of Genetics and Genomics, Boston University School of Medicine, 80 E. Concord St., Boston, Massachusetts 02118,¹ Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan,² Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan³

Received 15 November 2005/ Returned for modification 21 December 2005/ Accepted 14 February 2006

We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Pol to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Pol 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 Pol. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Pol. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Pol in a DNA damage-independent manner. Therefore, association of Pol 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 Pol. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Pol to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

X.B. and L.R.B. contributed equally to this work.

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