Structural and mechanistic studies Of DNA mismatch repair

大肠杆菌中的错配修复（MMR）是由三种蛋白MutS、MutL和MutH特异性地针对新合成的子链启动的。MutS是一种atp酶，可以识别不匹配的碱基对以及一条链上1-4个核苷酸的插入或删除。MutH是一种潜在的核酸内切酶，具有序列特异性和甲基化特异性；当检测到不匹配时被MutS激活时，它在半甲基化双工中将5剪切到未甲基化d（GATC）序列上。MutL也是一种atp酶，它介导MutS和MutH之间的通信，但两者并不直接相互作用。一旦一个缺口被MutH引入子链，UvrD解旋酶、单链结合蛋白和DNA外切酶被MutS和MutL招募，从缺口中去除核苷酸以超越错配。MutS和MutL的同源物存在于所有真核生物中，人类MutS或MutL同源物的功能障碍与遗传性非息肉病性结直肠癌（HNPCC）和其他散发性癌症的易感性直接相关。我们之前的研究确定了MutS的晶体结构，mts -错配DNA和mts -错配- adp复合物，MutL的N端和c端结构域，最后确定了MutH和MutH-DNA复合物。我们还鉴定了MutS和MutL atp酶的作用以及MutH的切割特异性。在本财政年度，我们已经成功地确定了UvrD解旋酶-DNA复合物的一系列晶体结构，这代表了UvrD在ATP水解循环中解开双链DNA的连续物理步骤。此外，我们还进行了诱变研究，以剖析UvrD解绕DNA的两种替代机制。我们的论文UvrD解旋酶通过两部分功率冲程一次解开DNA的一个碱基对，发表在2006年12月的《细胞》杂志上。目前，我们正在从事(1)获得大型蛋白质- dna组装体，例如MutL-DNA， MutL-UvrD-DNA， MutS-MutL-DNA复合物，用于结构表征；(2)参与错配修复的atp酶的预稳态动力学研究；(3)将结构和机制研究扩展到真核错配修复系统。在之前（2007-08），当李宰荣博士在NIH工作6年后返回韩国时，我们经历了一名员工的流失。徐婷博士将继续这项工作，他于2008年8月刚到这里继续这个项目。我们今年没有新的发表，但是真核错配修复项目已经取得了进展。

Mismatch repair (MMR) in E. coli is initiated by three proteins, MutS, MutL and MutH to specifically target newly synthesized daughter strand. MutS is an ATPase and recognizes a mismatched base-pair as well as an insertion or deletion of 1-4 nucleotides in one strand. MutH is a latent endonuclease that is both sequence- and methylation-specific; when activated by MutS upon detection of a mismatch, it cleaves 5 to the unmethylated d(GATC) sequence in a hemimethylated duplex. MutL is also an ATPase and mediates the communication between MutS and MutH, which do not directly interact. Once a nick is introduced to the daughter strand by MutH, UvrD helicase, single-strand binding protein and DNA exonuclease are recruited by MutS and MutL to remove nucleotides from the nick to beyond the mismatch. Homologues of MutS and MutL are found in all eukaryotes, and malfunction of either human MutS or MutL homolog is directly implicated in the susceptibility to hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. Our previous studies led to the determination of crystal structures of MutS, MutS-mismatch DNA and MutS-mismatch-ADP complexes, the N- and C-terminal domain of MutL, and finally MutH and MutH-DNA complexes. We also characterized the role of the MutS and MutL ATPases and the cleavage specificity of MutH. In this fiscal year, we have succeeded in determining a series of crystal structures of UvrD helicase-DNA complexes, which represent consecutive physical steps of UvrD unwinding a duplex DNA in an ATP hydrolysis cycle. In addition, we have carried out mutagenesis studies to dissect two alternative mechanisms of DNA unwinding by UvrD. Our manuscript UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke was published in Cell in December 2006. Currently we are engaging in (1) obtaining large protein-DNA asssemblies, e.g. MutL-DNA, MutL-UvrD-DNA, MutS-MutL-DNA complexes, for structural characterization, (2) pre-steady state kinetic studies of the ATPases involved in mismatch repair, and (3) expanding structural and mechanistic studies to eukaryotic mismatch repair systems. In the previous (2007-08), we experienced a staff lost when Dr. Jae Young Lee returned to Korea after a six year stint at NIH. Dr. Ting Xu, who is going to continue the work, has just arrived in Aug. 2008 to carry on the project. We don't have new publication this year, but progresses have been made in the eukaryotic mismatch repair project. References Lee, J. Y. and Yang, W. (2006). UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell, 127, 1349-1360. Yang, W. (2007). Human MuLa: the jack of all trades in mismatch repair is also an endonuclease. DNA Repair, 6(1) 135-9.