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Molecular and Cellular Biology, November 2005, p. 9350-9359, Vol. 25, No. 21
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.21.9350-9359.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Nuclear Dynamics of PCNA in DNA Replication and Repair

Jeroen Essers,1* Arjan F. Theil,1 Céline Baldeyron,1 Wiggert A. van Cappellen,2 Adriaan B. Houtsmuller,3 Roland Kanaar,1,4 and Wim Vermeulen1

Department of Cell Biology & Genetics,1 Department of Reproduction & Development,2 Department of Pathology,3 Department of Radiation Oncology, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands4

Received 26 May 2005/ Returned for modification 28 June 2005/ Accepted 22 July 2005

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

* Corresponding author. Mailing address: Dept. of Cell Biology and Genetics, Erasmus MC, Dr. Molenwaterplein 50, PO Box 1738, Rotterdam 2015GE, The Netherlands. Phone: 31104087158. Fax: 31104089468. E-mail: j.essers{at}erasmusmc.nl.

Molecular and Cellular Biology, November 2005, p. 9350-9359, Vol. 25, No. 21
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.21.9350-9359.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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