DNA SYNTHESIS--REGULATION IN NORMAL AND CANCER CELLS

DNA 合成——正常细胞和癌细胞中的调节

• 批准号: 2086324
• 负责人: EARL F BARIL
• 金额: $ 40.8万
• 依托单位: WORCESTER FOUNDATION FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2086324
• 关键词: DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; DNA topoisomerases; HeLa cells; cell cycle; cell growth regulation; chromatography; conformation; enzyme complex; enzyme mechanism; enzyme reconstitution; enzyme structure; genetic regulation; immunofluorescence technique; laboratory mouse; laboratory rabbit; neoplasm /cancer genetics; neoplastic cell; pancreatic ribonuclease; protein purification; simian virus 40; tumor antigens; virus DNA

The 21 S complex has been purified over 2,000-fold and the partially purified enzyme complex contains in addition to SV40 DNA in vitro replication activity; a 640 kDa multiprotein DNA polymerase alpha-primase complex, a dA/dT sequence binding origin recognition protein, DNA-dependent ATPase, DNA ligase I, histone H1 preferring protein kinase, proliferating cell nuclear antigen (PCNA), topoisomerase I and RNase H. The specific aims of the proposed project are the following. (1) To extensively purify the 21 S enzyme using conventional purification procedures, partially disassemble the complex by chromatography through coupled columns of native and denatured DNA-celluloses followed by anion exchange chromatography and purify the separated components. Reconstitution of the enzyme complex from its resolved components will be attempted. The steps of initiation, leading and lagging strand synthesis and supercoiling during T-antigen dependent replication of SV40 DNA in vitro will be investigated using the purified 21 S enzyme complex, reconstituted complex and purified subassemblies of the enzyme complex. (2) To determine the level of activity and organization of the 21 S complex of enzymes for DNA synthesis during the transition from G, to S and S to G2 phases of the cell cycle in synchronized HeLa cells in culture. (3) To investigate by immunofluorescent imaging analysis, using a variety of antibodies to components of the 21 S enzyme complex, the intracellular localization of the enzyme. The overall aim of the proposed project is to define possible controls for DNA biosynthesis during normal and abnormal growth. The immediate goal is to define the organization and control of the enzymatic machinery for chromosomal DNA replication in human cells. Our approach to this objective is to use simian virus 40 (SV40) DNA as a model replicon to study the human cell enzymatic machinery that participates in the initiation of T-antigen dependent replication of plasmid DNAs containing inserts of SV40 DNA containing sequence of the origin for replication. This approach led to the isolation from HeLa cell extracts of a 21 S megadalton complex of enzymes for DNA synthesis. The enzyme complex contains all of the activity for T-antigen-dependent and SV40 origin specific initiation of SV40 DNA replication in vitro.

21 S复合物已纯化超过2，000倍， 纯化的酶复合物在体外除含有SV 40 DNA外， 复制活性;一种640 kDa多蛋白DNA聚合酶α-引发酶 复合物，dA/dT序列结合起始识别蛋白，DNA依赖性 ATP酶，DNA连接酶I，组蛋白H1偏爱蛋白激酶，增殖 细胞核抗原（PCNA）、拓扑异构酶I和RNase H。 具体 拟议项目的目标如下。 (1)广泛净化 21 S酶使用常规纯化程序，部分 通过色谱法通过天然的偶联柱分解复合物 和变性DNA-纤维素，然后进行阴离子交换层析， 纯化分离的组分。 从酶复合物的重构 将尝试其解析的组件。 开始的步骤， T抗原过程中的前导链和滞后链合成以及超螺旋 SV 40 DNA的体外依赖性复制将使用 纯化的21 S酶复合物、重构的复合物和纯化的 酶复合物的降解。 (2)为了确定 DNA合成酶的21 S复合物的活性和组织 在细胞周期从G1到S和从S到G2期的过渡期间， 培养中的同步化HeLa细胞。 (3)调查 免疫荧光成像分析，使用各种抗体， 21 S酶复合物的组成部分， 酶 拟议项目的总体目标是确定可能的控制措施， 正常和异常生长期间的DNA生物合成。 近期目标是 定义酶机制的组织和控制， 人类细胞中的染色体DNA复制。 我们实现这一目标的方法 是使用猿猴病毒40（SV 40）DNA作为模型复制子来研究人类 参与T抗原起始的细胞酶机制 含有SV 40 DNA插入片段的质粒DNA的依赖性复制 含有复制起点的序列。 这种做法导致 从HeLa细胞提取物中分离21 S兆道尔顿复合物， DNA合成酶。 酶复合物包含了所有的活性 对于SV 40 DNA的T抗原依赖性和SV 40来源特异性启动 体外复制。

项目成果

Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.

从 HeLa 细胞的 DNA 引物酶-聚合酶 α 复合物中分离和纯化游离引物酶活性。

• DOI: 10.1093/nar/14.21.8467
• 发表时间: 1986
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Vishwanatha,JK;Baril,EF
• 通讯作者: Baril,EF

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

选择性抑制复制 DNA 合成和复制特异性 DNA 聚合酶的 N2-芳氨基嘌呤的设计和表征：对哺乳动物 DNA 聚合酶 α 和细菌 DNA 聚合酶 III 具有活性的鸟嘌呤衍生物。

• DOI: 10.1093/nar/10.14.4431
• 发表时间: 1982
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Wright,GE;Baril,EF;Brown,VM;Brown,NC
• 通讯作者: Brown,NC

Priming of DNA synthesis by diadenosine 5',5"'-P1,P4-tetraphosphate with a double-stranded octadecamer as a template and DNA polymerase alpha.

• DOI: 10.1073/pnas.79.6.1791
• 发表时间: 1982-03
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: P. Zamecnik;E. Rapaport;E. Baril

Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots.

哺乳动物 α 聚合酶：部分互补 DNA 的克隆和粗匀浆蛋白印迹中催化亚基的免疫结合。

• DOI: 10.1021/bi00377a041
• 发表时间: 1987
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: SenGupta,DN;Kumar,P;Zmudzka,BZ;Coughlin,S;Vishwanatha,JK;Robey,FA;Parrott,C;Wilson,SH
• 通讯作者: Wilson,SH

Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase.

来自 HeLa 细胞的单链 DNA 依赖性 ATP 酶可刺激 DNA 聚合酶 α-引物酶活性：ATP 酶的纯化和表征。

• DOI: 10.1021/bi00489a036
• 发表时间: 1990
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Vishwanatha,JK;Baril,EF
• 通讯作者: Baril,EF