Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1
Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1
复制标题
DOI：
10.1073/pnas.0505949102
复制
发表时间：
2005-12-20
影响因子：
11.1
通讯作者：
Burgers, PM
中科院分区：
文献类型：
--
作者：
Garg, P;Burgers, PM
在应对DNA损伤时，Rad6/Rad18泛素结合复合物在增殖细胞核抗原（PCNA）的赖氨酸 - 164位点进行单泛素化修饰。尽管PCNA的泛素化被认为是启动复制后修复的关键步骤，但是这种修饰的机制相关性仍然难以捉摸。在此，我们描述了一个强大的体外系统，该系统可特异性地在赖氨酸 - 164位点对酵母PCNA进行泛素化。重要的是，只有那些通过其加载因子复制因子C恰当地加载在效应DNA周围的PCNA钳才会被泛素化。这一观察结果表明，在体外，只有在停滞的复制叉处存在的PCNA才会被泛素化。泛素化的PCNA显示出与未修饰的PCNA相同的复制功能。这些功能包括通过复制因子C加载到DNA上，以及通过DNA聚合酶δ、 flap核酸内切酶FEN1和DNA连接酶I在PCNA协调作用下进行冈崎片段的合成和成熟。然而，尽管DNA聚合酶δ的活性不受PCNA泛素化的影响，但泛素化的PCNA特异性地激活了跨损伤合成中的两种关键酶：DNA聚合酶η（酵母中的着色性干皮病同源物）和Rev1（一种在组织诱变性DNA复制机制中起作用的脱氧胞苷酰转移酶）。我们提出PCNA的泛素化增加了其作为滑动钳的功能，以促进诱变性DNA复制。
In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase 8, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase C remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.