Nuclear dynamics of PCNA in DNA replication and repair
Nuclear dynamics of PCNA in DNA replication and repair
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DOI：
10.1128/mcb.25.21.9350-9359.2005
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发表时间：
2005-11-01
影响因子：
5.3
通讯作者：
Vermeulen, W
中科院分区：
文献类型：
--
作者：
Essers, J;Theil, AF;Vermeulen, W
DNA 聚合酶持续因子增殖细胞核抗原 (PCNA) 对于 DNA 复制和修复至关重要。环形同源三聚体 PCNA 环绕并沿着双链 DNA 滑动，充当将蛋白质定位到 DNA 的“滑动夹”。我们确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参与。复制灶的光漂白和追踪揭示了两个 PCNA 动力学池之间的动态平衡，即与复制灶结合并作为自由移动部分。为了同时监测 PCNA 在 DNA 复制和修复中的作用，我们局部造成了紫外线诱导的 DNA 损伤。令人惊讶的是，PCNA 在受损区域的停留时间比在复制灶的停留时间长。使用 DNA 修复突变体，我们发现 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均观察到 PCNA 在受损区域的局部积累，但在早期 S 期暂时消失。 S 期后期离散病灶中 PCNA 积累的重新出现可能反映了 PCNA 参与处理停滞复制叉的不同基因组维护过程，例如跨损伤合成 (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复制因子的从头组装引起的，并表明 PCNA 在 DNA 复制和修复中具有不同的结合亲和力。
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.