Jeon Yongmoon, Kim Daehyung, Martín-López Juana V, Lee Ryanggeun, Oh Jungsic, Hanne Jeungphill, Fishel Richard, Lee Jong-Bong
Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang, 790-784, Korea;
Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210;
Proc Natl Acad Sci U S A. 2016 Mar 22;113(12):3281-6. doi: 10.1073/pnas.1523748113. Epub 2016 Mar 7.
Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.
错配修复(MMR)由进化上保守的MutS同源物(MSH)和MutL同源物(MLH/PMS)激活。MSH识别错配的核苷酸并形成极其稳定的滑动夹,这些滑动夹可能被MLH/PMS结合,最终授权从远处的3'-或5'-DNA断裂处开始进行链特异性切除。与完整MMR反应相关的机械过程仍然是个谜。纯化的人(智人或Hs)5'-MMR切除反应需要HsMSH2-HsMSH6异二聚体、5'→3'核酸外切酶HsEXOI和单链结合异源三聚体HsRPA。HsMLH1-HsPMS2异二聚体在细胞提取物中对5'-MMR切除有显著影响,但在纯化系统中不是必需的。通过实时单分子成像,我们表明HsRPA或大肠杆菌EcSSB限制了HsEXOI对有切口或缺口的DNA的切除活性。HsMSH2-HsMSH6通过克服HsRPA/EcSSB的抑制作用来激活HsEXOI,并利用多个动态滑动夹增加切除片段长度。相反,HsMLH1-HsPMS2通过控制切除复合物的数量来调节切除片段长度,从而与5' MMR建立联系。