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.Proper duplication, maintenance, and repair of the genome are essential for ensuring genomic stability. Defects in any of these processes contribute to the onset and progression of cancer (9), because genomic DNA is subject to damage by both environmental agents and endogenous metabolic processes. In addition, stalling of the DNA replication machinery, which occurs upon encountering damaged DNA, is a challenging problem for cells. A variety of DNA damage bypass and repair mechanisms rescue replication after encountering DNA lesions. Protein clamps on DNA have been implicated in all processes of DNA metabolism, including DNA replication and repair. The most extensively investigated protein clamp in eukaryotes is the DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA).PCNA is a central protein in both DNA replication and repair. PCNA encircles double-stranded DNA as a trimer, forming a sliding clamp that tethers proteins such as polymerases to DNA (5). PCNA is essential not only for DNA replication but also for several forms of DNA repair, including nucleotide excision repair (NER), the major pathway by which cells remove DNA damage introduced by UV light and a variety of chemical carcinogens (10). After recognition of the lesion, the damaged strand is excised and resynthesized in a process requiring PCNA. ...