Structural Study Of DNA Recombination, Repair, Replicat

DNA 重组、修复、复制的结构研究

• 批准号: 7152617
• 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/7152617
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA methylation; DNA repair; DNA replication; Escherichia coli; T cell receptor; X ray crystallography; adenosinetriphosphatase; bacterial genetics; bacterial proteins; cell cycle; crystallization; enzyme activity; gene mutation; gene rearrangement; genetic recombination; hydrolysis; intermolecular interaction; nuclease; nucleic acid structure; protein biosynthesis; protein structure; protein structure function; structural biology

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 (MMR) 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. MutL 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, UvrD are recruited 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 suggest that the broad range of mismatch-repair substrates and high repair specificity are achieved with the high energy factor, ATP, utilized by MutS to verify and proofread mismatch recognition and to recruit MutL to signal for repair. In this year, we have determined the crystal structure of the C-terminal dimerization domain of MutL, characterized its DNA-binding and protein-interacting role in MMR. Based on out biochemical and genetic data, we propose a model that explains how the strand nicking (1st step) occurs either 5' or 3' to the mismatch site and the strand removal (2nd step by UvrD and exonucleases) is usually directed towards the mismatch site. In the past year, we have determined the crystal structures of MutH-DNA complexes with either unmethylated or hemimethylated DNA. We have further characterized how MutL activates MutH and UvrD and how metal ions influences substrate specificity. To understand the relationship between mismatch repair and DNA replication, we have solved the crystal structure of SeqA, a negative regulartor for replication initiation in E. coli and share the same DAN bidning site with MutH. Previously we determined the crystal structure of the DNA-binding domain complexed with its binding site, hemimethyalated GATC. This year, we solve the structure of the polymerization domain of SeqA, which allows us to build a functional SeqA polymer that accounts for its role in DNA replication and segregation. We propose that the competition between the two may regulate the mismatch repair specificity. 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 dimers. 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 eta 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 and a serier of crystal structures of Dpo4 complexed with a cyclobutane pyrimidine dimers, benzo[a]pyrene adduct, and abasic lesion in 2003 and 2004, this year we cap our studies of Dpo4 by determining its substrate specificity and nucleotide selection. Our structural and biochemical studies suggest that both replicative and translesion DNA polymerase depend on precise metal-ion coordination for the rate-limiting step ? the chemical bond formation. Along the line of metal ion and substate specificity, we have determined the crystal structures of RNase H complexed with an RNA/DNA hybrid substrate. RNase H is an essential enzyme for HIV replication and is the founding member of a large number of endonuclease families. Our crystal structures illustrate how the enzyme recognizes a specific substrate and the metal-dependent hydrolysis mechanism. Based on our structures, we are able to propose a general mechanism for nucleotidyl transfer reactions including DNA transposition and RNAi processing.

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的未甲基化d（GATC）序列连接。MutL调解MutS和MutH之间的通信，两者不直接交互。一旦通过MutH、UvrD解旋酶、单链结合蛋白和DNA核酸外切酶将切口引入子链，UvrD被募集以从切口去除核苷酸以超过错配。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。我们以前的研究表明，广泛的错配修复底物和高修复特异性是通过高能量因子ATP来实现的，ATP被MutS用来验证和校正错配识别，并招募MutL来发出修复信号。在这一年中，我们已经确定了MutL的C-末端二聚化结构域的晶体结构，表征了其在MMR中的DNA结合和蛋白质相互作用的作用。基于我们的生物化学和遗传数据，我们提出了一个模型，解释了链切口（第一步）如何发生在错配位点的5'或3'端，而链去除（第二步通过UvrD和核酸外切酶）通常指向错配位点。在过去的一年里，我们已经确定了MutH-DNA复合物的晶体结构与非甲基化或半甲基化的DNA。我们进一步表征了MutL如何激活MutH和UvrD以及金属离子如何影响底物特异性。 为了了解错配修复和DNA复制之间的关系，我们已经解决了SeqA的晶体结构，SeqA是一种在E. coli中表达，与MutH具有相同的DAN竞争位点。以前，我们确定了与其结合位点，半甲基化GATC复合的DNA结合结构域的晶体结构。今年，我们解决了SeqA聚合结构域的结构，这使我们能够构建一种功能性SeqA聚合物，解释其在DNA复制和分离中的作用。我们认为，两者之间的竞争可能会调节错配修复的特异性。 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 eta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，以及2003年和2004年发表了一系列Dpo 4与环丁烷嘧啶二聚体、苯并[a]芘加合物和无碱基损伤复合的晶体结构之后，今年我们通过确定其底物特异性和核苷酸选择来结束我们对Dpo 4的研究。我们的结构和生物化学研究表明，复制和translesion DNA聚合酶依赖于精确的金属离子协调的限速步骤？化学键的形成。 沿着金属离子和底物特异性的路线，我们测定了RNA/DNA杂交底物复合物RNase H的晶体结构。RNA酶H是HIV复制的必需酶，并且是大量核酸内切酶家族的创始成员。我们的晶体结构说明了酶如何识别特定的底物和金属依赖性水解机制。基于我们的结构，我们能够提出一个通用的机制，包括DNA转座和RNAi加工的核苷酸转移反应。