DNA-POLYMERASES DELTA AND EPSILON ARE REQUIRED FOR CHROMOSOMAL REPLICATION IN SACCHAROMYCES-CEREVISIAE
DNA-POLYMERASES DELTA AND EPSILON ARE REQUIRED FOR CHROMOSOMAL REPLICATION IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1128/mcb.13.1.496
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发表时间：
1993-01-01
影响因子：
5.3
通讯作者：
CAMPBELL, JL
中科院分区：
文献类型：
--
作者：
BUDD, ME;CAMPBELL, JL
三种DNA聚合酶，α，δ和β，是酿酒酵母中生存所必需的。我们已经研究了DNA聚合酶β和δ是否是DNA复制所必需的。两个温度敏感的突变，在POL2基因，编码DNA聚合酶的DNA，已被确定通过使用质粒洗牌技术。突变株中DNA合成产物的碱性蔗糖梯度分析表明，在异步培养转移到非允许温度后，没有形成染色体大小的DNA。观察到的唯一DNA合成是短DNA片段的量减少。来自这些突变体的复制中间体的DNA谱与在相同条件下在DNA聚合酶α缺陷的突变体中合成的DNA所观察到的那些相似。在DNA聚合酶α和DNA聚合酶β缺陷菌株中，DNA复制在转变到非允许温度时停止的发现表明两种DNA聚合酶都参与延伸。相比之下，以前的研究pol3突变体，缺乏DNA聚合酶δ，表明有相当大的残留DNA合成在非允许的温度。我们重新研究了pol3突变体中DNA合成的性质。我们发现，pol3菌株是有缺陷的染色体大小的DNA的合成在限制性温度释放后，从一个羟基脲块。这些结果表明，酵母DNA聚合酶δ也需要在复制叉。
Three DNA polymerases, alpha, delta, and epsilon, are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.