Base excision and DNA binding activities of human alkyladenine DNA glycosylase are sensitive to the base paired with a lesion
Base excision and DNA binding activities of human alkyladenine DNA glycosylase are sensitive to the base paired with a lesion
复制标题
DOI：
10.1074/jbc.m010641200
复制
发表时间：
2001-04-20
影响因子：
4.8
通讯作者：
Bloom, LB
中科院分区：
文献类型：
--
作者：
Abner, CW;Lau, AY;Bloom, LB
人烷基腺嘌呤DNA糖基酶具有广泛的底物特异性，可以从DNA中去除一组结构不同的受损嘌呤。为了更清楚地确定人烷基腺嘌呤DNA糖基酶底物专一性的结构和机制基础，在相同的DNA序列背景下，测量了几种不同损伤和未损伤的嘌呤的切割动力学和DNA结合活性。我们发现，1，N-6-亚乙基腺嘌呤(EpsilonA)和次黄嘌呤(HX)的切割效率较高，而7，8-二氢-8-氧鸟嘌呤、O-6-甲基鸟嘌呤、腺嘌呤和鸟嘌呤的切割效率较低。HX和epsilonA与T的切割的单周转动力学表明，HX的切割速度大约是epsilonA的4倍，而结合分析表明，epsilonA的结合亲和力大约是FIX的5倍，相反的嘧啶碱基对HX的切割动力学和DNA结合亲和力有显著影响，而对EA的影响较小。令人惊讶的是，用与HX相反的U取代T，使切除率显著降低15倍，亲和力增加7-8倍，人烷基腺嘌呤DNA糖基酶与含有碱性位点的DNA产物的结合亲和力与HX损伤的亲和力相似。
The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that 1,N-6-ethenoadenine (epsilonA) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O-6-methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and epsilonA paired with T showed that excision of Hx was about four times faster than epsilonA whereas binding assays showed that the binding affinity was about five times greater for epsilonA than for fix, The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for EA. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7-8, The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.