ENZYMATIC MECHANISMS OF DNA SYNTHESIS

哺乳动物新生DNA片段成熟为全长染色体 DNA正在使用纯化的小牛酶和合成的 引物模板起始RNA的去除可以通过RNase H1进行， 它特异性地切割掉完整的RNA引物，留下一个 连接到DNA片段的单核糖核苷酸。最后一个核苷酸是 被5'到3'核酸外切酶去除，该核酸外切酶通过从 上游DNA片段。产生一个缺口，由DNA连接酶密封 I.提出实验来澄清是否建议的序列 事件是体内的通路。由DNA聚合酶产生的RNA引发的DNA 在体内，α/引发酶将作为RNA酶H1的底物进行测试。的 上述模型提出，这些引物，无论序列如何， 高效特异地切割。核酸外切酶的能力， 将评估降解RNA引物的核酸酶，因为该核酸酶可以 替代RNase H。DNA聚合酶的身份， 参与冈崎片段处理的是未知的。核 将比较聚合酶α、δ和β，以及 重要的辅助蛋白RF-C、RP-A和PCNA，用于有效工作的能力 用两种核酸酶和DNA连接酶I，完成成熟 反应 5'至3'核酸外切酶也具有独特的核酸内切能力。它 可以从模板上的引物上切下未退火的5 ′片段， 存在紧邻的上游引物。这表明 启动子RNA可以通过核酸内切酶切去除， 上游引物被延长。DNA解旋酶E促进从 引物的5'末端，并将评估其促进 核酸内切引物去除。核酸外切酶似乎沿着 未退火的5'区，以便内切核酸裂解。一 核酸内切酶的可能目的是切过一个受损的片段， 然而，聚合物，它的运动可能会被某些类型的损坏所阻止。 测试其切割经过顺铂和更大修饰位点的能力 加合物，将不仅表明其在体内的功能，但将澄清其 作用机制。核酸内切酶功能产生 还将探索用于重组的底物。 将产生针对RNA酶H1（5'至3'核酸外切酶）和DNA的抗体。 解旋酶E.这些将用于免疫亲和纯化这些 蛋白质，检查其细胞周期调节，并测试其 细胞功能的抗体注射到青蛙卵母细胞。的同源物 核酸酶和解旋酶将在S.酿酒厂， 基因将被破坏以评估细胞作用。这种彻底的方法 应该可以确定冈崎片段成熟的机制。

Maturation of mammalian nascent DNA fragments to full length chromosomal DNA is being investigated using purified calf enzymes and synthetic primer-templates. Removal of initiator RNA can be carried out by RNase H1, which specifically cleaves off the intact RNA primer, leaving a monoribonucleotide attached to the DNA segment. This last nucleotide is removed by a 5' to 3' exonuclease, that is stimulated by synthesis from the upstream DNA segment. A nick is generated that is sealed by DNA ligase I. Experiments are proposed to clarify whether the proposed sequence of events is the pathway in vivo. RNA primed DNA made by DNA polymerase alpha/primase, as in vivo, will be tested as a substrate for RNase H1. The above model proposes that these primers, regardless of sequence, be cleaved efficiently and specifically. The ability of the exonuclease to degrade the RNA primer will be evaluated, since that nuclease could substitute for the RNase H. The identity of the DNA polymerase that participates in Okazaki fragment processing is not known. Nuclear polymerases alpha, delta and epsilon will be compared, together with the important co-proteins RF-C, RP-A and PCNA, for ability to work efficiently with the two nucleases and DNA ligase I, to complete the maturation reaction. The 5' to 3' exonuclease also has a unique endonucleolytic capacity. It can cut off an unannealed 5' segment from a primer on a template, only if there is an immediately adjacent upstream primer. This suggests that the initiator RNA could be removed by endonucleolytic cleavage, as the upstream primer is elongated. DNA helicase E promotes displacement from the 5' end of primers, and will be evaluated for ability to promote endonucleolytic primer removal. The exonuclease appears to thread along the unannealed 5' region in order to cleave endonucleolytically. One likely purpose of the endonuclease is to cut past a damaged segment of polymer, however, its motion could be blocked by some types of damage. Tests of its ability to cut past sites modified by cis-platinum and larger adducts, will not only suggest its function in vivo, but will clarify its mechanism of action. The capacity of the endonuclease function to generate substrates for recombination will also be explored. Antibodies will be generated to RNase H1, the 5' to 3' exonuclease and DNA helicase E. These will be used for immunoaffinity purification of these proteins, examination of their cell cycle regulation, and tests of their cellular function by antibody injection into frog oocytes. Homologs of the nucleases and helicase will be identified in S. cerevisiae, where the genes will be disrupted to evaluate cellular role. This thorough approach should define the mechanism of Okazaki fragment maturation.