MECHANISMS OF DNA-PROTEIN INTERACTION IN DNA REPLICATION

DNA 复制中 DNA-蛋白质相互作用的机制

• 批准号: 2178192
• 负责人: SUBHASIS B BISWAS
• 金额: $ 18.05万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178192
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; Saccharomyces cerevisiae; adenosinetriphosphatase; antibody; enzyme activity; enzyme mechanism; enzyme reconstitution; enzyme structure; exonuclease; gene expression; helicase; laboratory mouse; laboratory rabbit; nucleic acid sequence; polymerase chain reaction; protein purification; protein sequence; protein structure function

The overall goal of our research remains the investigation of the mechanism by which chromosomes replicate in eukaryotic cells using yeast as a model system. The progress made during the last two years of this project has defined our goals and our research is now centered on (i) multimeric DNA polymerase alpha with associated polymerase, primase, 5'->3'exonuclease, ssDNA dependent ATPase, and DNA helicase activities; (ii) a replication protein A (RP-A) dependent helicase; (iii) an origin binding protein that we have cloned. We have gained insight and isolated some of the proteins and enzymes that are likely involved in the initial stages of DNA replication. With regard to the multimeric DNA polymerase alpha, our goals are: (i) identification and isolation of the ATPase, helicase (helicase A), and 5'->3' exonuclease subunits, and characterization of their enzymatic activities; (ii) structural analysis of the polypeptides and preparation of polyclonal antibodies against each of these subunits; (iii) N-terminal amino acid sequencing, isolation, and characterization of genes for the exonuclease, ATPase, and helicase subunits. We plan to amplify and express the genes for the subunits and activities of multiprotein pol alpha complex with the following goals: (i) PCR amplify the genes for the multiprotein pol alpha subunits and carry out high level expression using E. coli or yeast expression vectors; (ii) structure function analysis of the expressed polypeptides; (iii) analyze the function(s) of the various subunits in DNA synthesis, processivity, and fidelity of the pol alpha complex. We have purified a 48 kDa novel RP-A dependent helicase (helicase B) to homogeneity. Our plan is to characterize the enzymatic activities of these helicases A & B and determine their roles in DNA replication. We have isolated the gene for an origin binding protein (OBP), that is distinct from the more abundant ARS binding factor I. Our present studies indicate that this protein exists as a part of a larger polypeptide complex. We have developed a tentative purification scheme for this complex. Our goals for deciphering the structure and function of this complex are as follows: (i) isolation of OBP complex in native form; (ii) identification and analysis of accessory proteins of OBP, if any; (iii) completion of sequencing and expression of the OBP.

我们研究的总体目标仍然是调查 利用酵母在真核细胞中复制染色体的机制 作为一个模型系统。 过去两年取得的进展 项目已经确定了我们的目标，我们的研究现在集中在（i） 多聚体DNA聚合酶α与相关聚合酶，引发酶， 5 '-> 3'核酸外切酶、ssDNA依赖性ATP酶和DNA解旋酶活性; (ii)复制蛋白A（RP-A）依赖性解旋酶;（iii）起点 我们克隆的结合蛋白。 我们已经获得了洞察力， 一些蛋白质和酶可能参与了最初的 DNA复制的阶段。 关于多聚体DNA聚合酶α，我们的目标是：（i） ATP酶、解旋酶（解旋酶A）和 5 '->3'外切核酸酶亚基，以及它们的酶促性质的表征 活性;（ii）多肽的结构分析和制备 针对这些亚基中的每一个的多克隆抗体;（iii）N-末端 氨基酸测序，分离和表征的基因， 外切核酸酶、ATP酶和解旋酶亚基。 我们计划扩大和 表达多蛋白聚合物亚基和活性的基因， （i）PCR扩增α复合物的基因， 多蛋白聚合酶α亚基，并进行高水平表达， E.大肠杆菌或酵母表达载体;（ii）结构功能分析 表达的多肽;（iii）分析各种多肽的功能， DNA合成中的亚基、持续合成能力和聚合酶α的保真度 复杂. 我们纯化了一个48 kDa的新的RP-A依赖性解旋酶 （解旋酶B）至均一。 我们的计划是将酶的特性 这些解旋酶A和B的活性，并确定它们在DNA中的作用 复制的 我们已经分离出了一种起源结合蛋白（OBP）的基因，即 不同于更丰富的ARS结合因子I。 我们目前 研究表明，这种蛋白质作为一种更大的 多肽复合物 我们已经制定了一个初步的净化方案 对于这个复杂的。 我们要破译的结构和功能 该复合物的分离方法如下：（i）天然OBP复合物的分离 形式;（ii）OBP的辅助蛋白的鉴定和分析，如果 （iii）完成OBP的测序和表达。