Francesconi S, Park H, Wang T S
Department of Pathology, Stanford University School of Medicine, CA 94305-5324.
Nucleic Acids Res. 1993 Aug 11;21(16):3821-8. doi: 10.1093/nar/21.16.3821.
DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.
DNA聚合酶α和δ是重要的酶，被认为在染色体DNA的起始和复制过程中发挥关键作用。在本研究中，我们发现粟酒裂殖酵母（S.pombe）DNA聚合酶α和δ（pol α+和pol δ+）的基因对于细胞活力至关重要。pol α+或pol δ+基因的破坏会导致不同的终末表型。粟酒裂殖酵母pol δ+基因能够在限制温度下互补酿酒酵母（S.cerevisiae）的温度敏感型cdc2-2等位基因。通过体外随机诱变，我们产生了三个pol δ条件致死等位基因。我们用诱变序列替换了pol δ+基因的野生型染色体拷贝，并在体内对温度敏感等位基因进行了表征。所有三个温度敏感突变体都表现出典型的细胞分裂周期（cdc）终末表型，类似于被破坏的pol δ+基因的表型。流式细胞术分析表明，在非允许温度下，所有三个突变体都停滞在细胞周期的S期。回收并测序了三个粟酒裂殖酵母条件性pol δ等位基因。导致温度敏感表型的突变是错义突变。改变的氨基酸残基在已知的聚合酶δ序列中是独特保守的。
