Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase.
Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase.
复制标题
研究 tyrosine-114 在人 O6-烷基鸟嘌呤-DNA 烷基转移酶活性中的作用。
作者：
Goodtzova,K;Kanugula,S;Edara,S;Pegg,AE
酪氨酸-114是O6-烷基鸟嘌呤-DNA烷基转移酶(AGT)所有已知序列中13个完全保守的氨基酸之一。通过将该氨基酸更改为人 AGT 中的苯丙氨酸 (F)、丙氨酸 (A)、苏氨酸 (T) 或谷氨酸 (E)，研究了该氨基酸在 AGT 修复烷基化 DNA 中的重要性。然后将突变蛋白的活性与野生型蛋白的活性进行比较，以执行以下操作：（a）保护大肠杆菌免受甲基化剂N-甲基-N'-硝基-N-亚硝基胍（MNNG）的侵害； (b) 体外修复甲基化DNA； (c)与含有O6-甲基鸟嘌呤的寡脱氧核苷酸结合； (d)与低分子量假底物O6-苄基鸟嘌呤反应。当在 E 中表达高水平时。缺乏内源性 AGT 的粘菌菌株 GWR109、野生型和 Y114F 突变体在减少突变和 MNNG 造成的细胞杀伤方面非常有效。 Y114A突变体的保护作用要小得多，突变体Y114T和Y114E则无活性。所有四种 AGT 突变体的纯化制剂显示与 O6-苄基鸟嘌呤反应速率的降低程度大致相似（74−120 倍）。相比之下，体外甲基化DNA底物活性降低的程度根据突变而变化，更保守的Y114F仅产生30倍的降低，而最剧烈的变化是Y114E完全消除了活性。改变Y114A导致活性降低1000倍，而Y114T则使活性降低10000倍。所有突变都会影响 AGT 与含有 O6-甲基鸟嘌呤的单链或双链寡脱氧核苷酸的结合。 Kd增加的程度根据氨基酸而变化，增加2-5倍(F)、7-11倍(A)、167-200倍(T)和600-1000倍(E)。这些结果与 tyrosine-114 在 AGT 与其 DNA 底物结合和促进烷基转移方面发挥的作用一致。 AGT 很可能与其他 DNA 修复蛋白类似，导致目标碱基从 DNA 螺旋中“翻转”出来。因此，Tyrosine-114 是在与翻转的 O6-甲基鸟嘌呤相互作用中发挥关键作用的优秀候选者。结果还表明，当细胞中产生大量 AGT 时，AGT 修复甲基化 DNA 的效率大幅下降并不会妨碍保护 E 的能力。来自有毒烷基化剂的大肠杆菌。突变体Y114F的活性降低了30倍，在带来这种保护方面与野生型AGT相同。
Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences ofO6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protectEscherichia colifrom the methylating agentN-methyl-N‘-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containingO6-methylguanine; and (d) react with the low molecular weight pseudosubstrate,O6-benzylguanine. When expressed at high levels inE. colistrain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74−120-fold) of reduction in the rate of reaction withO6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containingO6-methylguanine. The extent of increase in theKdvaried according to the amino acid with 2−5-fold (F), 7−11-fold (A), 167−200-fold (T), and 600−1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a “flipping out” of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flippedO6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protectE. colifrom toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.