Regulation of polymerase exchange between Polη and Polδ by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme

doi:10.1073/pnas.0801310105

Regulation of polymerase exchange between Polη and Polδ by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme

*Department of Chemistry, 414 Wartik Laboratory, Pennsylvania State University, University Park, PA 16802;
^‡Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1061; and
^§Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Temesvari krt.62, H-6726, Hungary

Contributed by Stephen J. Benkovic, February 11, 2008 (received for review January 9, 2008)

Abstract

To ensure efficient and timely replication of genomic DNA, organisms in all three kingdoms of life possess specialized translesion DNA synthesis (TLS) polymerases (Pols) that tolerate various types of DNA lesions. It has been proposed that an exchange between the replicative DNA Pol and the TLS Pol at the site of DNA damage enables lesion bypass to occur. However, to date the molecular mechanism underlying this process is not fully understood. In this study, we demonstrated in a reconstituted system that the exchange of Saccharomyces cerevisiae Polδ with Polη requires both the stalling of the holoenzyme and the monoubiquitination of proliferating cell nuclear antigen (PCNA). A moving Polδ holoenzyme is refractory to the incoming Polη. Furthermore, we showed that the Polη C-terminal PCNA-interacting protein motif is required for the exchange process. We also demonstrated that the second exchange step to bring back Polδ is prohibited when Lys-164 of PCNA is monoubiquitinated. Thus the removal of the ubiquitin moiety from PCNA is likely required for the reverse exchange step after the lesion bypass synthesis by Polη.

Footnotes

^†To whom correspondence may be sent at the present address:
Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE 19716.
E-mail: zzhuang{at}udel.edu
^¶To whom correspondence may be addressed: E-mail: sjb1{at}psu.edu
Author contributions: Z.Z. designed research; Z.Z. performed research; R.E.J., L.H., and S.P. contributed new reagents/analytic tools; Z.Z., R.E.J., L.H., L.P., S.P., and S.J.B. analyzed data; and Z.Z., L.H., S.P., and S.J.B. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0801310105/DCSupplemental.