Structural and mechanistic studies Of DNA mismatch repair

DNA错配修复的结构和机制研究

• 批准号: 7593542
• 负责人: WEI YANG
• 金额: $ 31.63万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593542
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Base Pair Mismatch; Base Pairing; Binding Proteins; C-terminal; Cells; Cleaved cell; Colorectal Cancer; Communication; Complex; DNA; DNA Repair; Daughter; Detection; Escherichia coli; Eukaryota; Eukaryotic Cell; Goals; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Human; Kinetics; Malignant Neoplasms; Manuscripts; Mediating; Methylation; Mismatch Repair; Molecular; Movement; Mutagenesis; Nucleotides; Power stroke; Predisposition; Proteins; Publishing; Recruitment Activity; Role; Series; Signal Transduction; Specificity; Structure; System; Time; cancer diagnosis; clinical application; endonuclease; exodeoxyribonuclease; helicase; interest; macromolecule

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.

错配修复(MMR)是由MutS、MutL和MuTH三种蛋白启动的，特异性地针对新合成的子链。MutS是一种ATPase，它识别不匹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，既是序列特异性的，也是甲基化特异性的；当被MutS检测到错配时，它会在半甲基化的双链中将5裂解为未甲基化的d(GATC)序列。MutL也是一种ATPase，介导MutS和Muth之间的通讯，而MutS和Muth之间不直接相互作用。一旦Muth将一个缺口引入到子链上，MutS和MutL就会招募UvrD解旋酶、单链结合蛋白和DNA外切酶来从缺口中去除核苷酸，从而超越错配。MutS和MutL的同源物存在于所有真核生物中，人类MutS或MutL同源物的故障直接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有关。我们以前的研究导致了MutS，MutS-错配DNA和MutS-错配-ADP复合体的晶体结构，MutL的N-末端和C-末端结构域，最后是MuTH和MuTH-DNA复合体。我们还研究了MutS和MutL ATPase的作用以及MUTH的切割特异性。在本财年，我们成功地确定了UvrD解旋酶-DNA复合体的一系列晶体结构，这些结构代表了UvrD在ATP水解循环中解开双链DNA的连续物理步骤。此外，我们还进行了突变研究，以剖析UvrD解开DNA的两种替代机制。我们的手稿UvrD解旋酶通过两个部分的力量一次一个碱基对地展开DNA，发表在2006年12月的《细胞》杂志上。目前，我们正致力于(1)获得大的蛋白质-DNA类化合物，如MutL-DNA、MutL-UvrD-DNA、MutS-MutL-DNA复合体，用于结构表征；(2)错配修复相关ATPase的稳态前动力学研究；(3)将结构和机制研究扩展到真核细胞的错配修复系统。 参考文献 李俊云和杨伟.(2006)。UvrD解旋酶通过两部分的力量冲程一次一个碱基对地解开DNA。电话：127,1349-1360。 杨伟.(2007)。人类木拉：错配修复的所有交易中的杰克也是一种核酸内切酶。DNA修复，6(1)135-9。