DNA POLYMERASES IN NORMAL AND NEOPLASTIC HUMAN CELLS

我们继续开发我们的单克隆抗DNA聚合酶α 抗体，以提供关于这种中心酶的新信息， 染色体复制 首先，使用可渗透的细胞系统， 建立了中和抗体抑制 人类细胞核中的复制DNA合成。 这表明中央 聚合酶α在染色体复制中的作用，并提供了一种新的 研究这种和其他DNA聚合酶在一种 多种复制和修复功能。 第二，利用我们的 免疫亲和纯化方案，我们已经积累了大量的 纯化的聚合酶全酶，现在正在获得氨基酸序列 作为第一种方法的几种催化活性肽的数据 进行基因分离实验 第三项成就是 成功鉴定一个基因位点是必要的， 足以表达人DNA聚合酶α活性， 定位于人类X染色体的短臂，在 带Xp21.3-Xp22.1。 通过调整这种方法，我们可以继续 确定偶联的DNA引发酶活性本身是否与 与所鉴定的遗传位点的关系，以及是否异源特异性亚基 在啮齿动物-人类体细胞杂交克隆中可检测到组装， DNA聚合酶α全酶复合物的产生。 信息 将从以下方面提供：1）非中和性单克隆抗体 区分人和啮齿动物聚合酶α肽的抗体;和 2)中和抗体与啮齿动物交叉反应的能力 聚合酶α组分，允许独立分离啮齿动物 和人全酶物种来自相同的啮齿动物-人杂交提取物。 第四，我们开发了一种免疫沉淀试验， 在体内一小时后鉴定聚合酶α肽 标记生长中的细胞。 分析表明，主要 翻译产物是多肽大家族中最大的成员， 也就是说，180 kDa的条带;因此该多肽家族的较小成员 可能由特定降解事件导致， 洁净. 现在有可能进行生理研究， 这种酶，例如，细胞周期中生物合成的时间， 细胞质和细胞核之间的易位，酶周转的动力学， 当细胞在循环和非循环之间通过时，对酶合成的控制 一个主要的目标将是分离聚合酶 α/引发酶基因序列，来自人cDNA和基因组文库。 （一）

We have continued to exploit our monoclonal, anti-DNA polymerase alpha antibodies to provide new information about this central enzyme of chromosomal replication. First, using a permeable cell system, we have established the capacity of the neutralizing antibodies to inhibit replicative DNA synthesis in human cell nuclei. This shows the central role of polymerase alpha in chromosomal replication and provides a novel approach to studies of the role of this and other DNA polymerases in a variety of replicative and repair functions. Second, by using our immunoaffinity purification protocol, we have accumulated quantities of the purified polymerase holoenzyme and are now obtaining amino acid sequence data on several of the catalytically active peptides as a first approach toward gene isolation experiments. A third accomplishment has been the successful identification of a single genetic locus that is necessary and sufficient for the expression of human DNA polymerase alpha activity and localizes to the short arm of the human X chromosome, at the junction of bands Xp21.3-Xp22.1. By adapting this methodology, we can proceed to establish whether the coupled DNA primase activity is itself tightly linked to the identified genetic locus, and whether heterospecific subunit assemblies are detectable in the rodent-human somatic cell hybrid clones in the generation of DNA polymerase alpha holoenzyme complexes. Information will be provided from: 1) the ability of the nonneutralizing monoclonal antibodies to discriminate human and rodent polymerase alpha peptides; and 2) the ability of the neutralizing antibodies to cross-react with rodent polymerase alpha fractions, permitting independent isolation of the rodent and human holoenzyme species from the same rodent-human hybrid extracts. Fourth, we have developed an immumoprecipitation assay that permits identification of the polymerase alpha peptide after one hour of in vivo labelling of growing cells. The assay indicates that the primary translation product is the largest member of the large polypeptide family, i.e., a band of l80kDa; thus the smaller members of this polypeptide family likely result from specific degradative events as a consequence of purification. It is now possible to carry out physiological studies on this enzyme, e.g., time of biosynthesis in cell cycle, kinetics of translocation between cytoplasm and nucleus, kinetics of enzyme turnover, control of enzyme synthesis as cells pass between cycling and noncycling states, etc. A major goal will be the isolation of polymerase alpha/primase genetic sequences from human cDNA and genomic libraries. (I)