Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase.
Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase.
复制标题
含有 O6-甲基鸟嘌呤类似物的寡脱氧核苷酸与野生型和突变型人 O6-烷基鸟嘌呤-DNA 烷基转移酶的反应和结合。
DOI：
10.1021/bi982908w
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Pegg,AE
中科院分区：
文献类型：
--
作者：
Spratt,TE;Wu,JD;Levy,DE;Kanugula,S;Pegg,AE
烷基鸟嘌呤-DNA烷基转移酶（AGT）通过将甲基从鸟嘌呤的6位转移到蛋白质上的半胱氨酸残基上来修复DNA。我们之前发现大肠杆菌蛋白与th6 -甲基鸟嘌呤（O6mG）在theN1-和do6位置发生关键的相互作用。人类AGT具有不同于细菌蛋白的特异性。我们将hAGT与含有o6mg类似物的双链十五脱氧核苷酸反应。二级速率常数依次为（×10-5M-1s-1）：O6mG（1.4）、o6 -甲基-6-硒鸟嘌呤（1.6）、> se6 -甲基-6-硒鸟嘌呤（0.1）、> s6 -甲基-6-硫鸟嘌呤（S6mG）（0.02）、s6 -甲基-6-硫代次黄嘌呤（S6mH）、o6 -甲基-1-去氮鸟嘌呤（O6m1DG）、o6 -甲基-3-去氮鸟嘌呤（O6m3DG）、do6 -甲基-7-去氮鸟嘌呤（O6m7DG）（均<0.0001）。电泳迁移率转移测定了其与hAGT的结合亲和力。含有ing6mg、S6mG和do6m3dg的寡脱氧核苷酸在竞争DNA存在时与AGT结合，Kd值在5 ~ 20 μM之间，而含有G、S6mH、O6m1DG和do6m7dg的寡脱氧核苷酸不与AGT结合（Kd值在200 μM以内）。这些结果表明，o6mg的1-、N2-和7-位点是与hAGT结合的关键位点，而3-和do6 -位点则参与了甲基转移。这些结果表明adaagt的活性位点比hAGT更灵活，这可能是adaagt比hAGT更快地与tho4mt反应的原因。
O6-Alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transferring the methyl group from the 6-position of guanine to a cysteine residue on the protein. We previously found that theEscherichia coliAda protein makes critical interactions withO6-methylguanine (O6mG) at theN1- andO6-positions. Human AGT has a different specificity than the bacterial protein. We reacted hAGT with double-stranded pentadecadeoxynucleotides containing analogues ofO6mG. The second-order rate constants were in the following order (×10-5M-1s-1):O6mG (1.4),O6-methylhypoxanthine (1.6) >Se6-methyl-6-selenoguanine (0.1) >S6-methyl-6-thioguanine (S6mG) (0.02) ≫S6-methyl-6-thiohypoxanthine (S6mH),O6-methyl-1-deazaguanine (O6m1DG),O6-methyl-3-deazaguanine (O6m3DG), andO6-methyl-7-deazaguanine (O6m7DG) (all <0.0001). Electrophoretic mobility shift assays were carried out to determine the binding affinity to hAGT. Oligodeoxynucleotides containingO6mG,S6mG andO6m3DG bound to AGT in the presence of competitor DNA withKdvalues from 5 to 20 μM, while those containing G,S6mH,O6m1DG, andO6m7DG did not (Kd> 200 μM). These results indicate that the 1-,N2-, and 7- positions ofO6mG are critical in binding to hAGT, while the 3- andO6-positions are involved in methyl transfer. These results suggest that the active site ofadaAGT is more flexible than hAGT and may be the reasonadaAGT reacts withO4mT faster than hAGT.