Wood Adam, Garg Parie, Burgers Peter M J
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
J Biol Chem. 2007 Jul 13;282(28):20256-63. doi: 10.1074/jbc.M702366200. Epub 2007 May 21.
During normal DNA replication, the proliferating cell nuclear antigen (PCNA) enhances the processivity of DNA polymerases at the replication fork. When DNA damage is encountered, PCNA is monoubiquitinated on Lys-164 by the Rad6-Rad18 complex as the initiating step of translesion synthesis. DNA damage bypass by the translesion synthesis polymerase Rev1 is enhanced by the presence of ubiquitinated PCNA. Here we have carried out a mutational analysis of Rev1, and we have identified the functional domain in the C terminus of Rev1 that mediates interactions with PCNA. We show that a unique motif within this domain binds the ubiquitin moiety of ubiquitinated PCNA. Point mutations within this ubiquitin-binding motif of Rev1 (L821A,P822A,I825A) abolish its functional interaction with ubiquitinated PCNA in vitro and strongly attenuate damage-induced mutagenesis in vivo. Taken together, these studies suggest a specific mechanism by which the interaction between Rev1 and ubiquitinated PCNA is stabilized during the DNA damage response.
在正常DNA复制过程中，增殖细胞核抗原（PCNA）可增强DNA聚合酶在复制叉处的持续合成能力。当遇到DNA损伤时，PCNA会被Rad6-Rad18复合物在赖氨酸164位点上进行单泛素化修饰，这是跨损伤合成的起始步骤。泛素化的PCNA的存在可增强跨损伤合成聚合酶Rev1对DNA损伤的跨越能力。在此，我们对Rev1进行了突变分析，并确定了Rev1 C末端中介导与PCNA相互作用的功能结构域。我们发现该结构域内的一个独特基序可结合泛素化PCNA的泛素部分。Rev1的这个泛素结合基序内的点突变（L821A、P822A、I825A）在体外消除了其与泛素化PCNA的功能相互作用，并在体内强烈减弱了损伤诱导的诱变作用。综上所述，这些研究提示了一种在DNA损伤应答过程中稳定Rev1与泛素化PCNA之间相互作用的特定机制。
