Study on Structure and Function of Mammalian DNA Replication Entyme

我们从tsFT 20细胞中分离出DNA聚合酶α催化亚基的cDNA，tsFT 20细胞是一种来自小鼠FM 3A细胞的温度敏感突变细胞系。DNA序列分析显示，该cDNA有一个单一的突变，胞嘧啶到胸腺嘧啶取代，改变氨基酸1180从丝氨酸到苯丙氨酸。此外，我们还在tsFT 20细胞的一个自发生长回复突变体和六个N-甲基-N '-硝基-N-亚硝基胍诱导的生长回复突变体中检测到突变位点。所有的回复突变细胞系都有第二个点突变邻近tsFT 20细胞中的第一个突变位点。在激活静止的瑞士小鼠3 T3细胞增殖期间，DNA聚合酶α-引发酶复合物的四个亚基的mRNA水平在DNA合成之前增加。为了分析这些基因的表达是如何控制的，我们分离了这些基因的上游区域并测定了它们的核苷酸序列。该复合物含有1-2个E_2F结合序列和10-14个DP回文序列，每个上游区域有一个AP_1结合基序，我们在大肠杆菌或昆虫细胞中过量表达了DNA聚合酶α-引发酶复合物的cDNA，纯化了基因产物，并制备了特异性抗体。共沉淀实验表明，54 K亚基与其他三种亚基都有亲和力。180 K亚基具有DNA聚合酶活性，46 K亚基具有引物酶活性。

We isolated cDNA of the catalytic subunit of DNA polymerase alpha from tsFT20 cells, a temperature-sensitive mutant cell line derived from mouse FM3A cells. DNA sequence analysis revealed that the cDNA has a single mutation, a cytosine to thymine substitution that changes amino acid 1180 from serine to phenylalanine. We have also shown that tsFT20 cells could be rescued by transfection with the wild-type cDNA.In addition, we detected mutation sites in one spontaneous and six N-methyl-N'-nitro-N-nitrosoguanidine-induced growth revertants of tsFT20 cells. All revertant cell lines had a second point mutation adjacent to the first mutation site in tsFT20 cells.During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In order to analyze how the expression of these genes are controlled, we have isolated upstream region of these genes and determined their nucleotide sequence. There are 1-2 E2F-binding sequence and 10-14 dp palindromic sequence, and one AP1-binding motif in each upstream region.We overexpressed cDNAs of the DNA polymerase alpha-primase complex in E.coli or insect cells, purified the gene products and raised the specific antibodies against them. Co-precipitation experiments showed that 54K subunit has affinity to all other three subunits. We have also shown that 180K subunit has DNA polymerase activity and 46K subunit has primase activity.