Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA
Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA
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DOI：
10.1093/nar/28.24.4912
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发表时间：
2000-12-15
影响因子：
14.9
通讯作者：
Yokota, J
中科院分区：
文献类型：
--
作者：
Shinmura, A;Yamaguchi, S;Yokota, J
腺嘌呤与8-羟基鸟嘌呤（OH（8）G）配对，是氧化DNA损伤的主要成分，在人类细胞中被MYH碱基切除修复蛋白切除。由于MYH蛋白对A：G错配的修复活性也已报道，我们比较了在草地贪夜蛾Sf 21细胞中表达的His（6）-标记的MYH蛋白对A：OH（8）G和A：OH（8）G的修复活性。G错配，比较线粒体1型蛋白和核2型蛋白的修复能力，以及通过大肠杆菌mutM mutY菌株的DNA切割测定和互补测定，将多态性1-Q（324）和2-Q（310）型蛋白与1-H（324）和2-H-310型蛋白进行比较。在低盐浓度（0-50 mM）的反应缓冲液中，在A：OH（8）G和A：G两种底物上检测到2型蛋白的腺嘌呤DNA糖基化酶活性。然而，在盐浓度为150 mM的反应缓冲液中，类似于生理条件，糖基化酶对A：G的活性而不是对A：oh（8）G的活性被极大地降低，并且2型蛋白对A：G的结合活性而不是对A：oh（8）G的结合活性成比例地降低。2型酶对A：OH（8）G的糖基化酶活性和抑制自发突变的能力大于1型酶。两种多态性MYH蛋白的修复活性无明显差异，表明在生理盐浓度下，人MYH蛋白特异性催化A：OH（8）G的糖基化反应。
Adenine paired with 8-hydroxyguanine (oh(8)G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells, Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His(6)-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh(8)G and A:G mismatches by DNA cleavage assay and gel mobility shift assay, We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q(324) and 2-Q(310) proteins with type 1-H(324)and 2-H-310 proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain, In a reaction buffer with a low salt (0-50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh(8)G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh(8)G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh(8)G, was proportionally reduced. The glycosylase activity on A:oh(8)G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins, These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh(8)G under physiological salt concentrations.