Characterization of Schizosaccharomyces pombe Hus1:: a PCNA-related protein that associates with Rad1 and Rad9
Characterization of Schizosaccharomyces pombe Hus1:: a PCNA-related protein that associates with Rad1 and Rad9
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DOI：
10.1128/mcb.20.4.1254-1262.2000
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发表时间：
2000-02-01
影响因子：
5.3
通讯作者：
Carr, AM
中科院分区：
文献类型：
--
作者：
Caspari, T;Dahlen, M;Carr, AM
Hus 1是所有裂殖酵母DNA完整性检查点所需的六个检查点Rad蛋白之一。MYC标记的Hus 1揭示了四种离散形式。主要形式，Hus 1-B，参与与Rad 9和Rad 1的蛋白复合物，与Rad 1-Hus 1免疫沉淀依赖于rad 9（+）位点的报道一致。一小部分Hu 1-B在未受损细胞中本质上被磷酸化，而更多的Hu 1-B在照射后被磷酸化。在用羟基脲阻断的早期S期细胞中，Hus 1-B磷酸化不增加，除非暴露时间延长。Rad 1-Rad 9-Hus 1-B复合物易于检测，但在可溶性提取物的共分级分离后，每种蛋白质的大部分不存在于该复合物中。间接免疫荧光显示，Hus 1是核，这种定位依赖于Rad 17。我们发现，Rad 17定义了一个独特的蛋白质复合物中的可溶性提取物，是从Rad 1，Rad 9，和Hus 1分开。然而，双杂交相互作用，在体外协会和在体内过表达实验表明Rad 1和Rad 17之间的瞬时相互作用。
Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9(+) locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1-Rad9-Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.