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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 {kappa} and Is Required for Recovery from S-Phase Checkpoint-Mediated Arrest

Xiaohui Bi,1,{dagger} Laura R. Barkley,1,{dagger} Damien M. Slater,1 Satoshi Tateishi,2 Masaru Yamaizumi,2 Haruo Ohmori,3 and Cyrus Vaziri1*

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

* Corresponding author. Mailing address: Department of Genetics and Genomics, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Phone: (617) 638-4175. Fax: (617) 414-1646. E-mail: cvaziri{at}bu.edu.

{dagger} X.B. and L.R.B. contributed equally to this work.

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.



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