MECHANISM OF DNA REPLICATION IN EUCARYOTES--YEAST AS A MODEL SYSTEM

酵母2 mum和ARS的体外DNA复制系统 （重复复制序列）质粒DNA， 几年前，这个实验室被用来研究 真核生物中DNA复制的机制 以识别 并纯化酵母DNA所需的酶和成分 复制，粗提物系统已分馏， 重组。 最近，又开展了两项活动， 鉴定和纯化：DNA拓扑异构酶I和ARS结合 蛋白质。 另一个研究小组独立地证明， DNA拓扑异构酶I是体内酵母DNA所必需的 复制和rRNA合成。 这符合我们 在体外观察，进一步加强我们的有用性， 体外系统 我们还表明，dnal-1突变体是 在染色体DNA复制的起始上有缺陷。 到 有助于过量生产和纯化DNA 1蛋白，DNA 1基因 已被克隆，其核苷酸序列已确定， 研究的规则。 最后，两个额外的单链DNA 从大肠杆菌中纯化了35 KD和45 KD的结合蛋白（SSB）， 酵母粗提物，进行了生物化学研究， 单特异性多克隆抗体从兔子中产生。 的 针对这些和先前纯化的SSB的抗体（14 KD，20 KD， 26 KD和38 KD）已经表明，38 KD和42 KD以及 20 KD和26 KD的SSBs在免疫学上是相关的，而14 KD的SSBs在免疫学上是相关的。 而35 KD的SSBs则为特有种。 这些抗体还 35 KD、38/42 KD和20 KD/26 KD单链抗体的定位 通过免疫荧光技术转移到细胞核。 35 KD SSB特别是 有趣和重要，因为它是公认的， 与哺乳动物PCNA/cyclin反应的抗血清，36 KD 一种受细胞周期控制的蛋白质，被认为与 细胞DNA复制

An in vitro DNA replication system of yeast 2 mum and ARS (autonomously-replicating-sequence) plasmid DNAs, developed in this laboratory several years ago, has been used to investigate the mechanism of DNA replication in eucaryotes. To identify and purify enzymes and components required for yeast DNA replication, the crude-extract system has been fractionated and reconstituted. Recently, two additional activities have been identified and purified: DNA topoisomerase I and ARS-binding protein(s). Independently, another group has shown that yeast DNA topoisomerase I is required for in vivo yeast DNA replication and for rRNA synthesis. This is consistent with our observation in vitro, further strengthening the usefulness of our in vitro system. We have also shown that the dnal-1 mutant is deficient in the initiation of chromosomal DNA replication. To aid in overproducing and purifying DNA1 protein, the DNA1 gene has been cloned, its nucleotide sequence determined and its regulation studied. Finally, two additional single-stranded DNA binding proteins (SSBs) (35KD and 45KD) have been purified from yeast crude extract, studied biochemically and their monospecific polyclonal antibodies raised from rabbits. The antibodies to these and to previously purified SSBs (14KD, 20KD, 26KD, and 38KD) have shown that the 38KD and 42KD and the 20KD and 26KD SSBs are immunologically related but the 14KD and 35KD SSBs are unique species. The antibodies have also been used to localize the 35KD, 38/42KD and 20KD/26KD SSBs to nuclei by immunofluoresence. The 35KD SSB is particularly interesting and important because it is recognized by an antiserum that reacts with mammalian PCNA/cyclin, a 36KD protein under cell-cycle control and thought to be involved in cellular DNA replication.