Isolation of yeast DNA replication mutants in permeabilized cells.
Isolation of yeast DNA replication mutants in permeabilized cells.
复制标题
在透化细胞中分离酵母 DNA 复制突变体。
DOI：
10.1073/pnas.80.21.6465
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发表时间：
1983
影响因子：
11.1
通讯作者：
Campbell,JL
中科院分区：
文献类型：
--
作者：
Kuo,C;Nuang,H;Campbell,JL
通过测定透化酵母DNA复制系统中DNA合成的缺陷来筛选温度敏感突变体的随机群体。已分离出20个体外DNA合成缺陷突变体。在本文中，我们描述了这些突变体中的八个。其中七个属于三个互补组cdc2，cdc8和cdc16，参与细胞分裂周期的控制。由于体外合成代表了在透化时体内活跃的复制叉的繁殖，我们发现cdc2和cdc16突变体可以在23 ℃而不是在37 ℃下将dTMP掺入这种透化细胞中的DNA中，这支持了这两种突变直接影响复制叉处DNA合成的结论。这种参与先前通过对CDC 2的体内分析提出，但对CDC 16不太清楚。最后，我们的筛选程序的有用性证明了在以前未描述的互补组的复制突变体的分离。一个菌株在体内DNA合成中表现出严重缺陷，但RNA合成正常。
A random population of temperature-sensitive mutants was screened by assaying for defects in DNA synthesis in a permeabilized yeast DNA replication system. Twenty mutants defective in in vitro DNA synthesis have been isolated. In this paper we describe eight of these mutants. Seven of them fall into three complementation groups--cdc2, cdc8, and cdc16--involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in vivo at the time of permeabilization, our finding that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23 degrees C but not at 37 degrees C supports the conclusion that these two mutations directly affect DNA synthesis at replication forks. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. Finally, the usefulness of our screening procedure is demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.