Structural Studies Of DNA Recombination, Repair, and Rep

DNA 重组、修复和重复的结构研究

• 批准号: 7337431
• 负责人: Yang Wei
• 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/7337431
• 关键词: Structural; Studies; DNA; Recombination; Repair

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 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 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 and biochemical characterization of the role of the MutS and MutL ATPases and the cleavage specificity of MutH. In the currrent year, we have succeeded in determining the crystal structure of UvrD helicase-DNA complexes, which represnt the consecutive physical steps of UvrD unwinding a duplex DNA in an ATP hydrolysis cycle (manuscript is being reviewed, Lee JY & Yang W). My group has sepnt nearly 4 years on the structural characterization of a 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. We and others found that the active site of Y-family polymerases are larger and more solvent exposed than that of replicative (high fidelity) polymerases, which explain their unique ability in translesion synthesis. A nagging question is why the Y-family polymerases have much better fidelity (accuracy in template-dependent DNA synthesis) than predicted from merely base:base hydrogen bonding. 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, last year we capped our studies of Dpo4 by determining its substrate specificity and nucleotide selection (Vaisman et al., 2005). 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. The latest study prompted us to take a close look on the two metal ions in the active site essential for the polymerase activity. In conjunction with our studies of sequence and structure-specific nucleases, MutH and RNase H, we conclude that the requirement of two Mg2+ ions for nucleic acid substrate binding and phosphoryl transfer reaction greatly enhances the catalytic specificity of the polymerases and nucleases (Nowotny & Yang, EMBO, 2006; Yang et al., Mol Cell, 2006). We are continuing our efforts in characterizing the role of RAG1 and RGA2 in V(D)J recombination. Most recently, our pursue has led down the path of studying histone modification as V(D)J recombination is dependent on transcription activation. RAG2 contains a PHD domain, which is recently shown to bind trimethylated Lys4 of histone H3. We have determined the crystal structures of the RAG2 PHD domain along and complexed with the modified H3 peptide. We are in the process of preparing the manuscript.

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介导MutS和MutH之间的通信，两者不直接相互作用。一旦通过MutH将切口引入子链，则募集UvrD解旋酶、单链结合蛋白和DNA核酸外切酶以将核苷酸从切口去除到超出错配。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和MutS和MutL ATP酶的作用和MutH的切割特异性的生化表征。近年来，我们成功地确定了UvrD解旋酶-DNA复合物的晶体结构，它代表了ATP水解循环中UvrD解旋双链DNA的连续物理步骤（手稿正在审查，Lee JY & Yang W）。 我的团队已经在Y家族DNA聚合酶的结构表征上工作了近4年，该聚合酶在未受损的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-oxo-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。我们和其他人发现Y家族聚合酶的活性位点比复制型（高保真）聚合酶的活性位点更大并且暴露更多的溶剂，这解释了它们在跨损伤合成中的独特能力。一个恼人的问题是为什么Y家族聚合酶具有比仅仅从碱基对碱基氢键预测的更好的保真度（模板依赖性DNA合成的准确性）。在2001年发表了第一个Y家族聚合酶和DNA复合物结构，并于2003年和2004年发表了一系列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Dpo 4晶体结构后，去年我们通过确定其底物特异性和核苷酸选择来完成了对Dpo 4的研究（Vaisman等人，2005年）。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。最新的研究促使我们仔细研究聚合酶活性所必需的活性位点中的两种金属离子。结合我们对序列和结构特异性核酸酶MutH和RNA酶H的研究，我们得出结论，核酸底物结合和磷酰基转移反应需要两个Mg 2+离子大大增强了聚合酶和核酸酶的催化特异性（Nowotny & Yang，EMBO，2006; Yang et al.，Mol Cell，2006）。 我们正在继续我们的努力，在V（D）J重组RAG 1和RGA 2的作用的特点。最近，我们的追求导致了研究组蛋白修饰的道路，因为V（D）J重组依赖于转录激活。RAG 2含有PHD结构域，其最近显示结合组蛋白H3的三甲基化Lys 4。我们已经确定了RAG 2 PHD结构域的晶体结构，沿着并与修饰的H3肽复合。我们正在准备手稿。