Methanococcus jannaschii flap endonuclease:: Expression, purification, and substrate requirements
Methanococcus jannaschii flap endonuclease:: Expression, purification, and substrate requirements
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DOI：
10.1128/jb.180.20.5406-5412.1998
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发表时间：
1998-10-01
影响因子：
3.2
通讯作者：
Wetmur, JG
中科院分区：
文献类型：
--
作者：
Rao, HGV;Rosenfeld, A;Wetmur, JG
皮瓣内切酶（FEN）的超嗜热古菌詹氏甲烷球菌在大肠杆菌中表达和纯化的同质性，FEN保留活性后，在95 ℃预孵育15分钟。一个假Y形基板形成的两个部分互补的寡核苷酸杂交。FEN切割具有邻近单链-双链体连接的游离5 '端的链。游离3 '端的缺失阻止了切割。互补寡核苷酸与游离3 '端的杂交使切割位点移动1至2个核苷酸。过量互补寡核苷酸与游离5 '端的杂交未能阻断切割，尽管该底物难以被Tag DNA聚合酶的5 '-3 '外切核酸酶活性切割。为了验证，用内部标记的发夹结构替换游离的5 '端。这种结构是FEN的底物，但只有在核酸外切切割使发夹不稳定后才成为Tag DNA聚合酶的底物。通过在病毒粒子phi X174上部分互补的寡核苷酸的引物延伸形成具有5 '单链分支的环状双链体底物。这种抗变性底物被用来检查温度和溶液性质的影响，如pH值，盐，和二价离子浓度上的营业额的酶。
The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity, FEN retained activity after preincubation at 95 degrees C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5 ' end adjacent to the single-strand-duplex junction. Deletion of the free 3 ' end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3 ' end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5 ' end failed to block cleavage, although this substrate was refractory to cleavage by the 5 '-3 ' exonuclease activity of Tag DNA polymerase. For verification, the free 5 ' end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Tag DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5 ' single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion phi X174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.