The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis

During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles.

在DNA复制过程中，错配修复蛋白（mismatch repair proteins）可识别并修复逃脱DNA聚合酶（DNA polymerase）校对活性的错配碱基（mispaired bases）。本研究针对“真核生物错配识别复合物（eukaryotic mismatch recognition complex）可随前进中的复制体（advancing replisome）行进”这一模型展开验证。我们以酵母为实验材料，利用参与错配识别的保守蛋白Msh2（Msh2）以及前导链聚合酶Polε（leading strand polymerase Polε）的亚基Pol2（Pol2），分别检测了复制过程中真核生物错配识别复合物与前导链聚合酶的动态变化。具体而言，我们对细胞进行同步化处理，并采用染色质免疫沉淀结合定制DNA平铺芯片（ChIP-chip）的方法对样本进行分析。G1期（G1）无法检测到Polε信号，但在S期（S-phase）全程的活性复制起始位点与正在复制的DNA区域均可观测到该信号。Polε信号可用于解析复制起始位点的激活时序、激活效率以及复制体推进速率。通过在同一菌株中同时检测Polε与Msh2的动态变化，我们证实错配识别复合物可结合复制起始位点，并随复制体扩散至邻近区域。在错配修复缺陷的增殖细胞核抗原（PCNA）突变体中，我们观测到Msh2仍可结合复制中的DNA区域，但其分布与动态变化发生了改变，这表明PCNA并非错配识别复合物结合复制区域的唯一决定因素，但可影响其移动的动态过程。我们结合生化与基因组学方法，证实两类MutS复合物（MutS complexes）均位于复制体附近，从而可在复制过程中高效修复全谱系的突变。本研究数据支持以下模型：MutSα/β（MutSα/β）可贴近复制体，从而在染色质重组装（chromatin reassembles）完成前高效修复新合成链上的突变。