Studies Of DNA Mismatch Repair

工作概述：本项目的目标是了解正常真核细胞中MMR基因的生物化学和遗传学，以及MMR基因突变如何导致环境相关的人类疾病。今年，我们证明了Mlh1-Pms1结合和水解ATP，并且它的两个ATP酶活性位点具有不同的结合亲和力和催化效率。我们发现，Mlh1-Pms1异源二聚体在Mlh1和Pms1 n端结构域中包含两个独立的DNA结合位点，并提供了初步证据，表明Mlh1的DNA结合对MMR功能很重要。我们发现Mlh1在生殖细胞发育中起作用，并初步了解了ATP结合、ATP水解和DNA结合在有丝分裂细胞和减数分裂细胞DNA交易中的重叠和/可分离作用。我们发现小鼠Exo1在突变消除中起作用，对雄性和雌性减数分裂至关重要。为了验证与复制滞后链模板相关的较低突变率是由于更有效地修复滞后链错配的假设，我们测量了ogg1菌株的突变率，这些菌株在3号染色体上复制起点相对两侧的位点上具有两个方向的报告等位基因。我们将MMR熟练菌株与MMR基因MSH2、MSH6、MLH1或EXOI缺失菌株进行了比较。MMR的缺失通过优先增加滞后链复制的诱变减少了链偏倚，表明8-O-G？后链复制过程中产生的错配更有效地修复。这与Okazaki片段和PCNA的5'端在滞后链复制过程中密度较高，被用作链识别信号的假设是一致的，从而为真核细胞体内该信号的同一性提供了第一个证据。在与Resnick小组的合作中，我们提供了镉抑制酵母和人类无细胞提取物中MMR活性的证据。

Summary of Work: The goals of this project are to understand the biochemistry and genetics of MMR in normal eukaryotic cells, and how mutations in MMR genes lead to environmentally associated human diseases. This year we demonstrated that Mlh1-Pms1 binds and hydrolyzes ATP, and that its two ATPase active sites have different binding affinity and catalytic efficiency. We showed that the Mlh1-Pms1 heterodimer contains two separate DNA binding sites in the Mlh1 and Pms1 N-terminal domains and provided initial evidence suggesting that DNA binding by Mlh1 is important for MMR function. We showed that Mlh1 functions in germ cell development, and provide initial insights into the overlapping and/ separable roles of ATP binding, ATP hydrolysis and DNA binding on DNA transactions in mitotic versus meiotic cells. We showed that mouse Exo1 functions in mutation voidance and is essential for male and female meiosis. To test the hypothesis that the lower mutagenesis associated with replicating lagging strand templates is due to more efficient repair of lagging stand mismatches, we measured mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compared a MMR proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1 or EXOI. Loss of MMR reduced the strand bias by preferentially increasing mutagenesis for lagging strand replication, indicating that 8-O-G?A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, both present at higher density during lagging strand replication, are used as strand discrimination signals, thus providing the first evidence for the identity of this signal in vivo in a eukaryotic cell. In collaboration with the Resnick group, we provide evidence that cadmium inhibits MMR activity in yeast and in human cell free extracts.