Structural and mechanistic studies Of DNA mismatch repair

E.大肠杆菌中，MutS、MutL和MutH三种蛋白质特异性地靶向新合成的子链。MutS是一种ATP酶，识别错配的碱基对以及一条链中1-4个核苷酸的插入或缺失。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;当检测到错配后被MutS激活时，它会切割半甲基化双链体中的未甲基化d（GATC）序列。MutL也是一种ATP酶，介导MutS和MutH之间的通讯，两者不直接相互作用。一旦MutH将切口引入子链，MutS和MutL就会募集UvrD解旋酶、单链结合蛋白和DNA核酸外切酶，以从切口中除去核苷酸，使其超出错配。MutS和MutL的同源物存在于所有真核生物中，并且人类MutS或MutL同源物的功能障碍直接涉及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。我们以前的研究导致MutS，MutS-错配DNA和MutS-错配-ADP复合物，MutL的N-和C-末端结构域，最后MutH和MutH-DNA复合物的晶体结构的确定。我们还表征了MutS和MutL ATP酶的作用以及MutH的切割特异性。在本财政年度，我们成功地确定了UvrD解旋酶-DNA复合物的一系列晶体结构，这些晶体结构代表了UvrD在ATP水解循环中解旋双链DNA的连续物理步骤。此外，我们还进行了诱变研究，以剖析两种替代机制的DNA解旋紫外线。我们的手稿UvrD解旋酶解旋DNA一个碱基对的时间由两部分的电力中风发表在细胞在2006年12月。目前，我们正在从事（1）获得大的蛋白质-DNA asssemblies，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酶的前稳态动力学研究，以及（3）将结构和机制研究扩展到真核错配修复系统。 引用 李，J. Y.和Yang，W.（2006年）。UvrD解旋酶通过两部分动力行程一次解旋一个碱基对。手机，127，1349-1360。 杨，W.（2007年）。人类穆拉：错配修复中的万能钥匙也是一种核酸内切酶。DNA修复，6（1）135-9。

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. 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.