MECHANISM OF DNA RECOMBINATION AND REPAIR IN YEAST SACCHAROMYCES CEREVISIAE

酿酒酵母 DNA 重组与修复机制

• 批准号: 3855937
• 负责人: A SUGINO
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3855937
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; Saccharomyces cerevisiae; fungal genetics; gene expression; genetic mapping; genetic recombination; molecular cloning; protein purification; protein sequence

It has been believed that DNA polymerase beta functions for DNA repair in mammalian cells. Although other DNA polymerase are well conserved functionally and structurally between yeast and mammalian cells, no beta-like DNA polymerase has been found in the yeast Saccharomyces cervisiae. We have identified and partially purified a new DNA polymerase. activity which is very similar to Drosophila melanogaster DNA polymerase beta. This polymerase is a basic protein, sensitive to dideoxylnucleotides, but not sensitive as the inhibitors which affect the activity of DNA polymerase alpha, delta, or epsilon, and its apparent MW is about 100 kDa. To further study this new DNA polymerase, we have been trying to clone gene for the DNA polymerase. REV3 gene, which is required for mutagenic DNA repair, encodes a polypeptide of ca. MW of 180 kDa and has several conserved regions specific for a-type DNA polymerases. However, it has not been established that the polypeptide is a DNA polymerase. In this year, we have expressed the REV3 gene product in insect cells using a vaccullo virus expression vector and have shown that the REV3 gene product has a DNA polymerase activity by using activity gel technique.

据信，DNA聚合酶β的功能是DNA修复， 哺乳动物细胞 虽然其他DNA聚合酶是很保守的 酵母和哺乳动物细胞之间的功能和结构，没有 在酵母菌中发现了一种β-样DNA聚合酶 cerebralae。 我们鉴定并部分纯化了一种新的DNA聚合酶。 活性与果蝇DNA聚合酶非常相似 β的 这种聚合酶是一种碱性蛋白质，对 双脱氧核苷酸，但不敏感的抑制剂，影响 DNA聚合酶α、δ或β的活性，其表观分子量为 约100 kDa。 为了进一步研究这种新的DNA聚合酶， 试图克隆DNA聚合酶的基因 REV 3基因是诱变DNA修复所需的基因，编码一个 的多肽。分子量为180 kDa，具有几个保守的区域特异性 a型DNA聚合酶。 然而，还没有确定， 多肽是DNA聚合酶。 今年，我们已经表达了REV 3 使用痘苗病毒表达载体在昆虫细胞中表达基因产物， 已经证明REV 3基因产物具有DNA聚合酶活性， 采用活性凝胶技术。