Nuclear dynamics of PCNA in DNA replication and repair.
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.
Essers
Jeroen
J
Dept. of Cell Biology and Genetics, Erasmus MC, Dr. Molenwaterplein 50, PO Box 1738, 2015 GE Rotterdam, The Netherlands. j.essers@erasmusmc.nl
Theil
Arjan F
AF
Baldeyron
Céline
C
van Cappellen
Wiggert A
WA
Houtsmuller
Adriaan B
AB
Kanaar
Roland
R
Vermeulen
Wim
W
D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle / Q000528:radiation effects; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009154:Mutation; D038761:Photobleaching; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D014466:Ultraviolet Rays
D018809:Proliferating Cell Nuclear Antigen; D049452:Green Fluorescent Proteins