Umar A, Buermeyer A B, Simon J A, Thomas D C, Clark A B, Liskay R M, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Cell. 1996 Oct 4;87(1):65-73. doi: 10.1016/s0092-8674(00)81323-9.
A two-hybrid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1. Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis. Activity was inhibited by p21WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions.
利用双杂交系统筛选酵母和人类表达文库，以寻找与错配修复蛋白相互作用的蛋白质。在两个文库中均回收得到增殖细胞核抗原（PCNA），且在酵母中显示其与MutL同源蛋白1（MLH1）和MutS同源蛋白2（MSH2）均相互作用。一个PCNA基因发生突变的酵母菌株在二核苷酸重复序列中具有显著升高的突变率，而在同时含有MLH1突变的菌株中该突变率并未进一步升高。使用一种不需要DNA修复合成的检测方法，对人类细胞提取物中的错配修复活性进行了检测。增殖抑制蛋白1（p21WAF1）或一种与PCNA结合的p21肽可抑制活性，而通过添加PCNA可使受抑制反应的活性恢复。数据表明，在DNA重新合成之前的步骤中，错配修复需要PCNA。PCNA与MLH1和MSH2结合的能力可能反映了错配修复与复制之间的联系，并且可能与错配修复蛋白在其他DNA事务中的作用相关。
