Masih Prerna Jasmine, Kunnev Dimiter, Melendy Thomas
Department of Cellular and Molecular Biology, Roswell Park Cancer Institute, Buffalo, NY 14214, USA.
Nucleic Acids Res. 2008 Jan;36(1):67-75. doi: 10.1093/nar/gkm943. Epub 2007 Nov 5.
Mismatch Repair (MMR) is closely linked to DNA replication; however, other than the role of the replicative sliding clamp (PCNA) in various MMR functions, the linkage between DNA replication and MMR has been difficult to investigate. Here we use an in vitro DNA replication system based on simian virus 40, to investigate MMR recruitment to replicating DNA. Both DNA replication and MMR proteins are recruited to replicating DNA in an origin-dependent fashion. Primer synthesis is required for recruitment of both PCNA and MMR proteins, but not for recruitment of the single-stranded DNA-binding protein (RPA). Blocking PCNA recruitment to replicating DNA with a p21-based polypeptide blocks PCNA and MMR, but not RPA recruitment. Once PCNA and subsequent proteins required for replication are loaded onto DNA, addition of p21 leaves PCNA on the replicating DNA, but actively displaces MMR proteins. These findings indicate that the MMR machinery is recruited to replicating DNA through its interaction with PCNA, and suggests that this occurs via binding of the MMR proteins to the multi-protein interaction sites on PCNA. These studies demonstrate the utility of this system for further investigation of the role of DNA replication in MMR.
错配修复（MMR）与DNA复制密切相关；然而，除了复制性滑动夹（PCNA）在各种MMR功能中的作用外，DNA复制与MMR之间的联系一直难以研究。在这里，我们使用基于猴病毒40的体外DNA复制系统，来研究MMR在复制DNA上的募集情况。DNA复制蛋白和MMR蛋白均以依赖于起始点的方式募集到复制DNA上。引物合成对于PCNA和MMR蛋白的募集是必需的，但对于单链DNA结合蛋白（RPA）的募集则不是必需的。用基于p21的多肽阻断PCNA募集到复制DNA上会阻断PCNA和MMR，但不会阻断RPA的募集。一旦PCNA和复制所需的后续蛋白加载到DNA上，添加p21会使PCNA留在复制DNA上，但会主动置换MMR蛋白。这些发现表明，MMR机制通过与PCNA的相互作用被募集到复制DNA上，并表明这是通过MMR蛋白与PCNA上的多蛋白相互作用位点结合而发生的。这些研究证明了该系统在进一步研究DNA复制在MMR中的作用方面的实用性。
