A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA
A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA
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DOI：
10.1093/nar/gki214
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Nakabeppu, Y
中科院分区：
文献类型：
--
作者：
Ushijima, Y;Tominaga, Y;Nakabeppu, Y
DATP氧化生成的2-羟基-2-脱氧腺苷三磷酸(2-OH-dATP)在DNA复制过程中会被模板DNA中鸟嘌呤相反的DNA聚合酶错掺入，从而导致自发突变。我们用纯化的重组硫氧还蛋白-mMUTYH融合蛋白证明了小鼠MUTYH(MMUTYH)具有DNA糖基化酶活性，不仅能去除腺嘌呤对8-氧鸟嘌呤(8-oxoG)，还能去掉2-羟基腺嘌呤(2-OH-A)对鸟嘌呤。MMUTYH与含腺嘌呤：8-oxoG对的双链寡核苷酸形成稳定的复合物，但几乎检测不到mMUTYH与含有2-OH-A：鸟嘌呤对的寡核苷酸的结合，这表明mMUTYH以不同的方式识别这两种底物并与其相互作用，这可能反映了每种底物的碱基切除修复过程的不同。突变的mMUTYH带有G365D氨基酸替换，对应于在家族性腺瘤性息肉病患者中发现的人类MUTYH的G382D胚系突变，它几乎完全保留了去除腺嘌呤而不是8-oxoG的DNA糖基酶活性，但具有去掉2-OH-A相反鸟嘌呤的1.5%的野生型活性。我们的结果表明，突变的hMUTYH(G382D)的修复能力降低，它低效地切除2-OH-A相反的鸟嘌呤，导致APC基因体细胞G：C到T：A颠倒突变的发生增加，以及结肠癌的发生。
2-Hydroxy-2-deoxyadenosine triphosphate (2-OH-dATP), generated by the oxidation of dATP, can be misincorporated by DNA polymerases opposite guanine in template DNA during DNA replication, thus causing spontaneous mutagenesis. We demonstrated that mouse MUTYH (mMUTYH) has a DNA glycosylase activity excising not only adenine opposite 8-oxoguanine (8-oxoG) but also 2-hydroxyadenine (2-OH-A) opposite guanine, using purified recombinant thioredoxin-mMUTYH fusion protein. mMUTYH formed a stable complex with duplex oligonucleotides containing an adenine:8-oxoG pair, but the binding of mMUTYH to oligonucleotides containing a 2-OH-A:guanine pair was barely detectable, thus suggesting that mMUTYH recognizes and interacts with these two substrates in a different manner which may reflect the difference in the base excision repair process for each substrate. Mutant mMUTYH with G365D amino acid substitution, corresponding to a G382D germline mutation of human MUTYH found in familial adenomatous polyposis patients, almost completely retained its DNA glycosylase activity excising adenine opposite 8-oxoG; however, it possessed 1.5% of the wild-type activity excising 2-OH-A opposite guanine. Our results imply that the reduced repair capacity of the mutant hMUTYH(G382D), which inefficiently excises 2-OH-A opposite guanine, results in an increased occurrence of somatic G:C to T:A transversion mutations in the APC gene as well as tumorigenesis in the colon.