The influence of the cdc27 subunit on the properties of the Schizosaccharomyces pombe DNA polymerase δ
The influence of the cdc27 subunit on the properties of the Schizosaccharomyces pombe DNA polymerase δ
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DOI：
10.1074/jbc.m202897200
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发表时间：
2002-09-27
影响因子：
4.8
通讯作者：
Hurwitz, J
中科院分区：
文献类型：
--
作者：
Bermudez, VP;MacNeill, SA;Hurwitz, J
裂殖酵母Pombe DNA聚合酶Delta包含Pol3、CDc1、CDc27和CDm1四个亚基。在这份报告中，我们研究了CDC27对polDelta的结构和活性的作用。我们证明了四亚基复合体在结构上是单体的，而不是以前的报道(Zuo，S.，Bermudez，V.，Zhang，G.，Kelman，Z.和Hurwitz，J.(2000)J.Biol Chem)。275、5153-5162)。早些时候和最近的观测结果之间的这种差异可以追溯到Cdc27明显的不对称形状。CDC27包含两个关键结构域，这两个结构域决定了它在激活polDelta中的作用。N-末端(氨基酸(AA)1-160)与CDc1结合，其末端(AA 362-369)与增殖细胞核抗原(PCNA)相互作用。与野生型复合体相比，S.pombe polDelta的突变体含有截短的CDc27衍生物，缺乏与增殖细胞核抗原的结合，支持DNA复制的过程较少。最小的增殖细胞核抗原结合基序(AA 352372)与C端截短的CDC27衍生物的融合恢复了体外过程中的脱氧核糖核酸合成。在体内，将这些融合的CDC27衍生物引入CDC27Delta细胞中，可以提高细胞的存活率。这些数据支持这样的模型，在该模型中，CDC27通过将增殖细胞核抗原招募到PolDelta型全酶中在DNA复制中发挥重要作用。
Schizosaccharomyces pombe DNA polymerase (pol) delta contains four subunits, pol 3, Cdc1, Cdc27, and Cdm1. In this report, we examined the role of Cdc27 on the structure and activity of pol delta. We show that the four-subunit complex is monomeric in structure, in contrast to the previous report that it was a dimer (Zuo, S., Bermudez, V., Zhang, G., Kelman, Z., and Hurwitz, J. (2000) J. Biol Chem. 275, 5153-5162). This discrepancy between the earlier and recent observations was traced to the marked asymmetric shape of Cdc27. Cdc27 contains two critical domains that govern its role in activating pol delta. The N-terminal region (amino acids (aa) 1-160) binds to Cdc1 and its extreme C-terminal end (aa 362-369) interacts with proliferating cell nuclear antigen (PCNA). Mutants of S. pombe pol delta, containing truncated Cdc27 derivatives deficient in binding to PCNA, supported DNA replication less processively than the wild-type complex. Fusion of a minimal PCNA-binding motif (aa 352372) to C-terminally truncated Cdc27 derivatives restored processive DNA synthesis in vitro. In vivo, the introduction of these fused Cdc27 derivatives into cdc27Delta cells conferred viability. These data support the model in which Cdc27 plays an essential role in DNA replication by recruiting PCNA to the pol delta holoenzyme.