DNA SYNTHESIS--REGULATION IN NORMAL AND CANCER CELLS

21号S建筑群已经被提纯了2000多倍，部分 纯化的酶复合体在体外除SV40 DNA外还含有 复制活性；640 kDa多蛋白DNA聚合酶α-引物 复合体，DNA依赖的da/dt序列结合的起源识别蛋白 ATPase，DNA连接酶I，组蛋白H1首选蛋白激酶，增殖 细胞核抗原、拓扑异构酶I和核糖核酸酶H 拟议项目的目标如下。(一)广泛净化 21 S酶采用常规纯化程序，部分 通过层析通过连接的天然柱来拆解该络合物 变性DNA-纤维素，然后阴离子交换层析和 对分离出的组分进行提纯。重组酶复合体的初步研究 将尝试其解析的组件。启蒙的步骤， T抗原合成中的超前和滞后链合成及超螺旋 SV40DNA在体外的依赖复制将使用 纯化21种S酶复合体、重组复合体及纯化 酶复合体的亚组分。(二)确定 21 S脱氧核糖核酸合成酶复合体的活性和组织 在细胞周期由G期过渡到S、S和G2期的过程中， 同步化培养的HeLa细胞。(三)以下列方式调查 免疫荧光成像分析，使用各种抗体来 21 S酶复合体的组成、胞内定位 这种酶。 拟议项目的总体目标是定义可能的控制 DNA在正常和异常生长过程中的生物合成。眼下的目标是 定义酶机械的组织和控制 人类细胞中的染色体DNA复制。我们实现这一目标的方法 是用猴病毒40(SV40)DNA作为模型复制子来研究人类 参与T抗原启动的细胞酶机制 含SV40DNA插入物的质粒DNA的依赖复制 包含复制起始点的序列。这种方法导致了 21个S百万吨级复合体HeLa细胞提取物的分离 用于DNA合成的酶。酶复合体包含了所有的活性 对于T抗原依赖和SV40起源的SV40 DNA的特异性启动 体外复制。

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.