RNase H2 of Saccharomyces cerevisiae is a complex of three proteins
RNase H2 of Saccharomyces cerevisiae is a complex of three proteins
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DOI：
10.1093/nar/gkh209
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Crouch, RJ
中科院分区：
文献类型：
--
作者：
Jeong, HS;Backlund, PS;Crouch, RJ
RNase H2的组成一直是一个长期存在的问题。细菌和古细菌的RNase H2作为单个多肽具有活性，而酿酒酵母的同源物Rnh2Ap在大肠杆菌中表达时不能产生活性的RNase H2。通过亲和层析纯化和鉴定与标记酵母Rnh2Ap相关的多肽，我们获得了RNase H2活性所必需的三个蛋白质复合物（Rnh2Ap， Ydr279p和Ylr154p）。编码任何一种蛋白质的基因的缺失或Rnh2A催化位点的突变都会导致RNase H2活性的丧失。即使当s.c reevisiae的RNase H2在催化上受到损害时，它仍然倾向于在模拟rna引物的Okazaki片段的底物中切割核糖核苷酸-脱氧核糖核苷酸序列5'侧的磷酸二酯键或嵌入双链DNA中的单个核糖核苷酸。有趣的是，Ydr279p和Ylr154p只有在近亲物种中才有同源蛋白。酿酒酵母RNase H2的多亚基性质对于结构目的和提供与其他参与DNA复制/修复和转录的蛋白质相互作用的手段可能是重要的。
The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins (Rnh2Ap, Ydr279p and Ylr154p) that together are necessary and sufficient for RNase H2 activity. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when S.cerevisiae RNase H2 is catalytically compromised, it still exhibits a preference for cleavage of the phosphodiester bond on the 5' side of a ribonucleotide-deoxyribonucleotide sequence in substrates mimicking RNA-primed Okazaki fragments or a single ribonucleotide embedded in a duplex DNA. Interestingly, Ydr279p and Ylr154p have homologous proteins only in closely related species. The multisubunit nature of S.cerevisiae RNase H2 may be important both for structural purposes and to provide a means of interacting with other proteins involved in DNA replication/repair and transcription.