THE B-SUBUNIT OF THE DNA-POLYMERASE ALPHA-PRIMASE COMPLEX IN SACCHAROMYCES-CEREVISIAE EXECUTES AN ESSENTIAL FUNCTION AT THE INITIAL-STAGE OF DNA-REPLICATION
THE B-SUBUNIT OF THE DNA-POLYMERASE ALPHA-PRIMASE COMPLEX IN SACCHAROMYCES-CEREVISIAE EXECUTES AN ESSENTIAL FUNCTION AT THE INITIAL-STAGE OF DNA-REPLICATION
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DOI：
10.1128/mcb.14.2.923
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发表时间：
1994-02-01
影响因子：
5.3
通讯作者：
PLEVANI, P
中科院分区：
文献类型：
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作者：
FOIANI, M;MARINI, F;PLEVANI, P
四亚基DNA聚合酶-引物酶复合物在从头合成DNA链的能力方面是独一无二的，一些体外数据表明它参与染色体DNA复制的起始和延伸，尽管在起始反应中起作用的直接体内证据仍然缺乏。该复合物的B亚基的功能尚不清楚，但编码该蛋白的酿酒酵母POL12基因对细胞活力至关重要。对克隆基因进行体外诱变，获得了不同的pol12等位基因。对18个pol12等位基因的体内分析表明，保守的羧基末端三分之二的蛋白质含有对细胞活力至关重要的区域，而更多样化的nh2末端部分是部分可缺性的。温度敏感的pol12-T9突变等位基因的特征表明，B亚基是体内DNA合成和通过S期正确进展所必需的。此外，互移实验表明，POL12基因产物在染色体DNA复制的早期阶段，在羟基脲敏感步骤之前起着至关重要的作用。提出了B亚基在起始DNA复制中的作用模型。
The four-subunit DNA polymerase cy-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.