ALTERATION OF ARGININE-128 TO ALANINE ABOLISHES THE ABILITY OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE TO REPAIR METHYLATED DNA BUT HAS NO EFFECT ON ITS REACTION WITH O-6-BENZYLGUANINE
ALTERATION OF ARGININE-128 TO ALANINE ABOLISHES THE ABILITY OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE TO REPAIR METHYLATED DNA BUT HAS NO EFFECT ON ITS REACTION WITH O-6-BENZYLGUANINE
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DOI：
10.1021/bi00021a024
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发表时间：
1995-05-30
期刊：
影响因子：
2.9
通讯作者：
PEGG, AE
中科院分区：
文献类型：
--
作者：
KANUGULA, S;GOODTZOVA, K;PEGG, AE
O-6-烷基鸟嘌呤-DNA烷基转移酶（AGT）是一种DNA修复蛋白，可从DNA中去除致突变的O-6-甲基鸟嘌呤损伤。为了获得更多的信息AGT的作用机制，在一个假定的DNA结合结构域中的两个保守残基被改变的定点突变，突变蛋白结合DNA，修复甲基化的DNA，并转换为鸟嘌呤O-6-苄基鸟嘌呤的能力进行了检查。精氨酸-128变为丙氨酸（R128 A）使AGT对甲基化DNA底物的活性降低了1000倍以上，但并没有降低与O-6-苄基鸟嘌呤的反应速率。在所使用的试验中，残基酪氨酸-114变为谷氨酸（Y114 E）完全消除了修复DNA中O-6-甲基鸟嘌呤的能力，表明其降低了> 15000倍，但将O-6-苄基鸟嘌呤转化为鸟嘌呤的能力仅降低了60倍。将该残基改变为丙氨酸（Y114 A）使对甲基化DNA的活性降低>1000倍，并且使对O-6-苄基鸟嘌呤的活性降低约80倍。无论是R128 A还是Y114 E突变体AGT都不能与对照AGT竞争甲基化DNA的修复，而半胱氨酸受体位点改变为丙氨酸的失活突变体C145 A在该测定中有效竞争。这些结果表明，精氨酸-128和酪氨酸-114残基参与AGT的DNA结合特性。AGT蛋白与DNA形成稳定复合物的能力因此通过测量电泳过程中DNA的阻滞来检验。突变体Y114 E不与单链或双链M13 DNA形成复合物，也不与单链或双链形式的寡脱氧核苷酸16聚体形成复合物。突变体R128 A确实与双链M13 DNA形成了良好的延迟复合物，但与单链M13 DNA或与单链16-mer不形成这样的复合物。一些复合物的形成与双链16聚体，但这是不太稳定的控制AGT形成的复合物。这些结果提供了直接的证据，AGT的结构域包含残基114和128参与DNA结合。突变体R128的结果进一步表明，在AGT反应期间产生单链区域，并且精氨酸-128参与以允许烷基转移发生的构象结合该单链区域。
O-6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes the promutagenic O-6-methylguanine lesion from DNA. In order to obtain more information about the mechanism of action of AGT, two conserved residues in a putative DNA binding domain were changed by site-directed mutagenesis, and the abilities of the mutant proteins to bind to DNA, to repair methylated DNA, and to convert O-6-benzylguanine to guanine were examined. The alteration of arginine-128 to alanine (R128A) reduced the AGT activity toward methylated DNA substrates by a factor of more than 1000 but did not decrease the rate of reaction with O-6-benzylguanine. The change of residue tyrosine-114 to glutamic acid (Y114E) completely abolished the ability to repair O-6-methylguanine in DNA in the assays used showing that this was reduced by >15 000-fold, but the ability to convert O-6-benzylguanine to guanine was reduced by only 60-fold. Alteration of this residue to alanine (Y114A) reduced activity toward methylated DNA by >1000-fold and toward O-6-benzylguanine by about 80-fold. Neither the R128A nor the Y114E mutant AGT were able to compete with the control AGT for the repair of methylated DNA whereas the inactive mutant, C145A, in which the cysteine acceptor site is changed to alanine, competed effectively in this assay. These results suggest that the residues arginine-128 and tyrosine-114 are involved in the DNA binding properties of the AGT. The ability of the AGT proteins to form stable complexes with DNA was therefore examined by measuring the retardation of DNA during electrophoresis. The mutant Y114E did not form complexes with either single-stranded or double-stranded M13 DNA or with an oligodeoxynucleotide 16-mer in a single-stranded or duplex form. Mutant R128A did form a well retarded complex with double-stranded M13 DNA but did not form such a complex with single-stranded M13 DNA or with the single-stranded 16-mer. Some complex formation occurred with the double-stranded 16-mer, but this was less stable than the complex formed by control AGT. These results provide direct evidence that the domain of the AGT containing residues 114 and 128 is involved in DNA binding. The results with the mutant R128 further suggest that a single-stranded region is generated during the AGT reaction and that arginine-128 is involved in binding this single-stranded region in a conformation that allows alkyl transfer to occur.