Kannouche Patricia, Lehmann Alan
Laboratory of Genetics Instability and Cancer, CNRS, Institut Gustav Roussy, Villejuif, France.
Methods Enzymol. 2006;408:407-15. doi: 10.1016/S0076-6879(06)08025-6.
During translesion synthesis past sites of damaged DNA, specialized Y-family polymerases are employed by the cell to replace the high stringency replicative polymerases and synthesize DNA past the damaged site. These polymerases are localized in replication factories during the S phase of the cell cycle. When progress of the replication fork is blocked, the polymerase accessory protein, proliferating cell nuclear antigen (PCNA), becomes ubiquitinated and the monoubiquitinated PCNA has an increased affinity for Y-family DNA polymerase eta (poleta). This chapter describes methods for visualizing the polymerases in replication factories, for analyzing the ubiquitination status of PCNA, and for measuring its interaction with poleta in chromatin extracts.
在跨越受损DNA位点进行跨损伤合成时，细胞会利用特殊的Y家族聚合酶取代高严谨性的复制性聚合酶，并在受损位点之后合成DNA。这些聚合酶在细胞周期的S期定位于复制工厂。当复制叉的进展受阻时，聚合酶辅助蛋白增殖细胞核抗原（PCNA）会发生泛素化，单泛素化的PCNA对Y家族DNA聚合酶η（polη）的亲和力会增加。本章介绍了在复制工厂中可视化这些聚合酶、分析PCNA泛素化状态以及测量其在染色质提取物中与polη相互作用的方法。
