Structural Studies Of DNA Recombination, Repair, and Rep

DNA 重组、修复和重复的结构研究

• 批准号: 6810307
• 负责人: WEI YANG
• 金额: --
• 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/6810307
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; T cell receptor; X ray crystallography; adenosinetriphosphatase; antibody; bacterial genetics; cell cycle; crystallization; enzyme activity; gene mutation; gene rearrangement; genetic recombination; hydrolysis; intermolecular interaction; nuclease; protein biosynthesis; protein structure function

DNA is susceptible to a variety of mutations and chemical modifications. Errors during DNA replication, either mispairing or slippage, result in mismatched base pairs, which occur at a frequency of 10-8 to 10-6. Exposure to UV irradiation or chemical agents may lead to covalently modified DNA bases, and programmed meiotic and mitotic DNA rearrangement, ionizing radiation and oxidative agents can result in double-strand DNA breaks. To maintain genomic integrity and to sustain life, bacteria, archaea and eukarya use conserved mechanisms to repair or to tolerate each type of damage. My research group has continued to carry on structural and functional studies of E. coli and human mismatch repair processes and lesion-bypass DNA synthesis. Mismatch repair in E. coli is initiated by three proteins, MutS, MutL and MutH, to specifically target the 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, thus targeting mismatch repair to newly synthesized and unmethylated daughter strand. MutL mediates the communication between MutS and MutH, which do not directly interact. Once a nick is introduced in the daughter strand by MutH, DNA exonuclease, UvrD helicase, single-strand binding protein and DNA polymerase III are recruited to remove nucleotides from the nick to beyond the mismatch and to fill in the resulting gap. 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. After determining the crystal structures of MutS, MutL and MutH between 1998 and 2001, we have constructed 47 structure-based mutations of MutS, MutL and MutH and analyzed the function of each protein in a series of in vitro assays including DNA binding, ATPase activity, DNA cleavage and mismatch repair initiation. In collaboration with Dr. Jeffrey Miller at UCLA, we have also characterized mutation and recombination rates of E. coli cells expressing the mutant proteins in vivo. Results from the mutant studies not only support the mismatch repair model we proposed that MutS stays bound to the mismatch site while activating the repair process but also illuminate the coordination between mismatch repair and prevention of homologous recombination between similar but not identical DNA sequences. Lesion-bypass DNA synthesis is carried out by the recently discovered Y-family DNA polymerases, which perform low-fidelity synthesis on undamaged DNA templates and are able to traverse normally replication-blocking lesions, including abasic sites, 8-oxo-G, benzopyrene adducts, and cyclobutane pyrimidine dimmers. Y-family polymerases are widespread and enable species from E. coli to human to tolerate UV irradiation and various forms of base modification. Each individual Y-family polymerase exhibits a distinct substrate preference. For example, Pol h is particularly efficient to bypass the UV crosslinking product, cyclobutane pyrimidine dimers. Mutations in XPV, which encodes human Pol h, are correlated to 20% of xeroderma pigmentosum. After publishing the first Y-family polymerase and DNA complex structure in 2001, we have recently determined the crystal structures of an archaeal Y ?family polymerase, Dpo4, complexed with a cyclobutane pyrimidine dimers. Our structures suggest a mechanism by which specific Y-family polymerases are able to bypass a thymine dimer while replicative DNA polymerases cannot. We are continuing to study structure of multiprotein and nucleic acid complexes involving in DNA repair and replication to elucidating molecular mechanism that underlies in human diseases.

DNA易受各种突变和化学修饰的影响。DNA复制过程中的错误，无论是错配还是滑动，都会导致碱基对错配，其发生频率为10-8至10-6。暴露于紫外线照射或化学试剂可导致共价修饰的DNA碱基，程序性减数分裂和有丝分裂DNA重排，电离辐射和氧化剂可导致双链DNA断裂。为了维持基因组的完整性和维持生命，细菌、古细菌和真核生物使用保守的机制来修复或耐受每种类型的损伤。本课题组继续对E.大肠杆菌和人类错配修复过程和病变旁路DNA合成。 E.大肠杆菌中，MutS、MutL和MutH三种蛋白质可以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，是序列和甲基化特异性，当激活MutS后检测到的错配，它裂解5？与半甲基化双链体中未甲基化的d（GATC）序列结合，从而将错配修复靶向新合成的未甲基化的子链。MutL介导MutS和MutH之间的通信，两者不直接相互作用。一旦通过MutH在子链中引入切口，DNA核酸外切酶、UvrD解旋酶、单链结合蛋白和DNA聚合酶III被募集以从切口去除核苷酸以超过错配并填充所得的缺口。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。在1998年至2001年确定MutS、MutL和MutH的晶体结构后，我们构建了47个基于结构的MutS、MutL和MutH突变，并在一系列体外试验中分析了每个蛋白的功能，包括DNA结合、ATP酶活性、DNA切割和错配修复启动。在与加州大学洛杉矶分校的Jeffrey米勒博士的合作中，我们也描述了大肠杆菌的突变和重组率。在体内表达突变蛋白的大肠杆菌细胞。突变体研究的结果不仅支持我们提出的错配修复模型，即MutS在激活修复过程的同时保持与错配位点的结合，而且还阐明了错配修复与防止相似但不相同的DNA序列之间同源重组之间的协调。 病变旁路DNA合成是由最近发现的Y家族DNA聚合酶进行的，其在未受损的DNA模板上进行低保真度合成，并且能够穿过正常的复制阻断病变，包括脱碱基位点、8-氧代-G、苯并芘加合物和环丁烷嘧啶二聚体。Y家族聚合酶广泛存在，并使来自E.大肠杆菌对人耐受紫外线照射和各种形式的碱基修饰。每个单独的Y家族聚合酶表现出不同的底物偏好。例如，Pol h对于绕过UV交联产物环丁烷嘧啶二聚体特别有效。编码人Pol h的XPV中的突变与20%的着色性干皮病相关。在2001年发表了第一个Y家族聚合酶和DNA复合物结构之后，我们最近确定了古细菌Y？家族聚合酶，Dpo 4，与环丁烷嘧啶二聚体复合。我们的结构表明一种机制，通过这种机制，特定的Y-家族聚合酶能够绕过胸腺嘧啶二聚体，而复制型DNA聚合酶不能。 我们将继续研究参与DNA修复和复制的多蛋白质和核酸复合物的结构，以阐明人类疾病的分子机制。