DNA SYNTHESIS: REGULATION IN NORMAL AND CANCER CELLS

这项研究的目的是确定可能的控制DNA 在正常和异常生长期间的生物合成。 一种多蛋白形式的DNA 聚合酶（聚合酶α2）已从 HeLa细胞核和细胞质的组合低盐提取物（0.15M KCl） 细胞 dna聚合酶α2 已被分解成催化剂， 通过疏水亲和和离子交换色谱进行亚基结构 和色谱聚焦。 它由DNA聚合酶α催化 核心酶（Mr = 180，000），"引发酶"（Mr = 70，000），5 '，5 ""二腺苷 四磷酸（Ap4A）结合蛋白（Mr = 92，000，47，000的二聚体）， 单链特异性核酸外切酶（Mr = 69，000），具有校对功能 功能和两个辅助蛋白Cl（Mr = 96，000， 24，000）和C2（Mr = 52，000）作为引物识别蛋白 在体外与引物单链DNA模板。 多克隆抗体 （兔）已针对均相DNA聚合酶α制备 催化核心亚基和Ap4结合蛋白 所述抗体是 用于研究多蛋白相互作用的拓扑结构 DNA聚合酶α亚单位2. 聚合酶α的抗体 和ap4A被用来研究DNA聚合酶的相互作用 阿尔法2 和ap4A在染色体复制中。 的蛋白质印迹分析 去除HeLa细胞匀浆和纯化的DNA聚合酶α2 与 聚合酶α催化核心亚基的抗体显示单一条带 183，000人。 所有的DNA聚合酶α2 可提取 0.15M KCl处理。 DNA聚合酶α核心亚基与C1、C2蛋白的相关性 用引物酶调控寡核苷酸引物的大小 这是合成的。 DNA聚合酶α-引物酶复合体合成RNA 在确定的DNA模板上的长度为7至15个核糖核苷酸的引物。 的 dna聚合酶α2 缔合引发酶合成五核苷酸 在相同的定义的DNA模板上的引物。 我们正在研究 DNA聚合酶α的相互作用2 和DNA的其他成分 SV40 DNA体外复制的合成机制。 （一）

The objective of this research is to define possible controls for DNA biosynthesis during normal and abnormal growth. A multiprotein form of DNA polymerase (polymerase alpha2) has been purified to homogeneity from the combined low-salt extract (0.15M KCl) of nuclei and cytoplasm of HeLa cells. DNA polymerase alpha2 has been resolved into its catalytic and subunit structure by hydrophobic affinity and ion exchange chromatography and chromatofocusing. It is composed of a DNA polymerase alpha catalytic core enzyme (Mr = 180,000), a "primase" (Mr = 70,000), 5',5''' diadenosine tetraphosphate (Ap4A) binding protein (Mr = 92,000, a dimer of 47,000), a single-strand specific exonuclease (Mr = 69,000) that has a proof-reading function in vitro and two accessory proteins Cl (Mr = 96,000, a tetramer of 24,000) and C2 (Mr = 52,000) that function as primer-recognition proteins in vitro with primed single-stranded DNA templates. Polyclonal antibodies (rabbit) have been prepared against the homogeneous DNA polymerase alpha catalytic core subunit and the Ap4A binding protein. The antibodies are being used to study the topology of the interactions of the multiprotein subunits of DNA polymerase alpha2. The antibodies to polymerase alpha and Ap4A are being used to study the interactions of DNA polymerase alpha2 and Ap4A in chromosome replication. Western blot analysis of erude HeLa cell homogenates and purified DNA polymerase alpha2 with the antibody to the polymerase alpha catalytic core subunit shows a single band of Mr 183,000. All of the DNA polymerase alpha2 is extractable from nuclei by 0.15M KCL. The association of DNA polymerase alpha core subunit and C1, C2 proteins with the primase appear to regulate the size of oligoribonucleotide primer that is synthesized. DNA polymerase alpha-primase complex synthesizes RNA primers of 7 to 15 ribonucleotides in legnth on defined DNA templates. The DNA polymerase alpha2 associated primase synthesizes pentanucleotide primers on the same defined DNA templates. We are studying the interactions of DNA polymerase alpha2 and other components of the DNA synthesizing machinery in the in vitro replication of the SV40 DNA. (I)