FISSION YEAST WITH DNA-POLYMERASE DELTA TEMPERATURE-SENSITIVE ALLELES EXHIBITS CELL-DIVISION CYCLE PHENOTYPE
FISSION YEAST WITH DNA-POLYMERASE DELTA TEMPERATURE-SENSITIVE ALLELES EXHIBITS CELL-DIVISION CYCLE PHENOTYPE
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DOI：
10.1093/nar/21.16.3821
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发表时间：
1993-08-11
影响因子：
14.9
通讯作者：
WANG, TSF
中科院分区：
文献类型：
--
作者：
FRANCESCONI, S;PARK, H;WANG, TSF
DNA聚合酶α和Delta被认为在染色体DNA的起始和复制中起着关键作用。在这项研究中，我们证明了裂殖酵母(S.pombe)DNA聚合酶α和Delta(polpha+和polDelta+)基因对细胞活力是必不可少的。Polpha+或polDelta+基因的中断会导致截然不同的末端表型。在有限的温度下，S.pombe polDelta+基因能够补充酿酒酵母(S.cerevisiae)的cdc2-2等位基因。通过体外随机诱变，我们产生了三个PolDelta条件致死等位基因。我们用突变后的序列替换了polDelta+基因的野生型染色体拷贝，并在体内对温度敏感的等位基因进行了表征。这三个温度敏感突变体都表现出典型的细胞分裂周期(CDC)末端表型，类似于中断的polDelta+基因。流式细胞仪分析显示，在不允许的温度下，三个突变体都被阻止在细胞周期的S期。回收了3个S.pombe条件等位基因，并对其进行了测序。导致温度敏感表型的突变是错义突变。在已知的聚合酶三角洲序列中，改变的氨基酸残基是唯一保守的。
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 (polalpha+ and poldelta+) are essential for cell viability. Disruption of either the polalpha+ or poldelta+ gene results in distinct terminal phenotypes. The S.pombe poldelta+ 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 poldelta conditional lethal alleles. We replaced the wild type chromosomal copy of poldelta+ 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 poldelta+ 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 poldelta 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.