Essers Jeroen, Theil Arjan F, Baldeyron Céline, van Cappellen Wiggert A, Houtsmuller Adriaan B, Kanaar Roland, Vermeulen Wim
Dept. of Cell Biology and Genetics, Erasmus MC, Dr. Molenwaterplein 50, PO Box 1738, 2015 GE Rotterdam, The Netherlands.
Mol Cell Biol. 2005 Nov;25(21):9350-9. doi: 10.1128/MCB.25.21.9350-9359.2005.
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.
DNA聚合酶持续合成因子增殖细胞核抗原（PCNA）对于DNA复制和修复都至关重要。环形同三聚体PCNA环绕双链DNA并沿其滑动，充当将蛋白质定位到DNA上的“滑动夹”。我们测定了绿色荧光蛋白标记的人PCNA（GFP-hPCNA）在活细胞中的行为，以分析其在DNA复制和修复中的不同作用。对复制位点进行光漂白和跟踪揭示了PCNA的两个动力学池之间的动态平衡，即与复制位点结合的以及作为自由移动部分的。为了同时监测PCNA在DNA复制和修复中的作用，我们局部施加紫外线诱导的DNA损伤。观察到PCNA在受损区域的停留时间比在复制位点长得多。使用DNA修复突变体，我们表明PCNA最初募集到受损位点依赖于核苷酸切除修复。在所有细胞周期阶段都观察到PCNA在受损区域的局部积累，但在S期早期暂时消失。PCNA在S期后期在离散位点重新积累可能反映了PCNA参与处理停滞复制叉的不同基因组维持过程，如跨损伤合成（TLS）。使用无法参与TLS的GFP-hPCNA泛素化突变体，我们注意到其在受损区域的停留时间明显缩短。我们的结果表明，PCNA位置的变化是由核质池中自由移动的复制因子重新组装引起的，并表明PCNA在DNA复制和修复中具有不同的结合亲和力。
