Characterization of Schizosaccharomyces pombe rad2 protein, a FEN-1 homolog
Characterization of Schizosaccharomyces pombe rad2 protein, a FEN-1 homolog
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DOI：
10.1093/nar/26.16.3645
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发表时间：
1998-08-15
影响因子：
14.9
通讯作者：
Doetsch, PW
中科院分区：
文献类型：
--
作者：
Alleva, JL;Doetsch, PW
在pombe Schizosaccharomyces pombe中发现了一个序列与fin -1蛋白编码基因rad2（+）相似的基因。我们报道了假设的S.pombe fin -1同源基因Rad2p的过表达、纯化和鉴定，一个GST-Rad2p融合蛋白在酿酒酵母中过表达，并通过GST亲和层析纯化到接近同源性。虽然Rad2p先前根据氨基酸同源性被归类为假定的fen1蛋白，但没有生化证据证明其具有皮瓣内切酶活性，但对几种不同寡脱氧核苷酸结构的DNA切割分析表明，GST-Rad2p具有5'-皮瓣内切酶和5‘- > - 3’双链DNA外切酶活性。GST-Rad2p在双链区分支点的一个碱基3‘处切割一个5’-flap和一个5'-伪y结构，并降解双链DNA。本文首次报道了S.pombe Rad2p的生化特性，并讨论了Rad2p在DNA切除修复和其他核酸反应中的潜在作用。
FEN-1 proteins are a family of nucleases essential for lagging strand DNA synthesis, A gene with sequence similarity to FEN-1 protein-encoding genes, rad2(+), has been identified in Schizosaccharomyces pombe, We report the overexpression, purification, and characterization of the putative S.pombe FEN-1 homolog, Rad2p, A GST-Rad2p fusion protein was overexpressed in Saccharomyces cerevisiae and purified to near homogeneity by GST affinity chromatography. Although Rad2p had been previously classified as a putative FEN-1 protein based on amino acid homology, there has been no biochemical evidence demonstrating flap endonuclease activity, DNA cleavage analysis of several different oligodeoxynucleotide structures indicates that GST-Rad2p possesses both 5'-flap endonuclease and 5'-->3' double-stranded DNA exonuclease activities. GST-Rad2p incises a 5'-flap and a 5'-pseudo-Y structure one base 3' of the branch point in the duplex region and also degrades double-stranded DNA, This is the first report on the biochemical characterization of S.pombe Rad2p, The potential roles of Rad2p in DNA excision repair and other nucleic acid reactions are discussed.