Substrate binding pocket residues of human alkyladenine-DNA glycosylase critical for methylating agent survival
Substrate binding pocket residues of human alkyladenine-DNA glycosylase critical for methylating agent survival
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DOI：
10.1016/j.dnarep.2008.06.019
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发表时间：
2008-10-01
期刊：
影响因子：
3.8
通讯作者：
Loeb, Lawrence A.
中科院分区：
文献类型：
--
作者：
Chen, Cheng-Yao;Guo, Haiwei H.;Loeb, Lawrence A.
人烷基腺嘌呤-DNA糖基酶（AAG）启动碱基切除修复（BER）的烷基化和脱胺碱基。在这里，我们评估了AAG底物结合袋的易变性，以及个体结合袋氨基酸对甲基化损伤存活的重要性。我们使用寡核苷酸定向诱变随机化19种氨基酸，其中8种与底物碱基相互作用，并创建了超过450万个变体。我们在修复缺陷大肠杆菌中表达了突变体AAGs，并选择了甲基甲烷磺酸盐（MMS）或甲基-lexitropsin （Me-lex）的细胞毒性保护，甲基-lexitropsin是一种产生3-甲基腺嘌呤作为主要碱基损伤的药物。对116个甲基化抗性突变体的序列分析显示，高度保守的Tyr（127）和His（136）没有被取代。相比之下，一个突变L180F在MMS-和me -lex抗性文库中都大量富集。在用于选择的高剂量MMS下，L180F单突变体的表达使存活率提高了4.4倍。均匀的L180F突变酶从甲基化的小牛胸腺DNA中显示出2.2倍的3-甲基腺嘌呤和7.3倍的7-甲基鸟嘌呤的切除。突变的糖基化酶减少了甲基化碱基的切除，可以促进高浓度MMS下的存活，在高浓度MMS下，下游酶处理有毒BER中间体的能力可能饱和。该突变体还显示，从寡核苷酸底物中分别减少6.6倍和3.0倍的1，n -6-乙烯腺嘌呤和次黄嘌呤的切除，与含碱基位点DNA的结合增加1.7倍。我们的工作为从晶体结构推断出的底物结合机制提供了体内证据，阐明了Leu（180）在野生型人类AAG中的功能，并与BER酶在损伤存活中的平衡表达作用相一致。(c) 2008 Elsevier B.V.版权所有
Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.5 million variants. We expressed the mutant AAGs in repair-deficient Escherichia coli and selected for protection against the cytotoxicity of either methylmethane sulfonate (MMS) or methyl-lexitropsin (Me-lex), an agent that produces 3-methyladenine as the predominant base lesion. Sequence analysis of 116 methylation-resistant mutants revealed no substitutions for highly conserved Tyr(127) and His(136). In contrast, one mutation, L180F, was greatly enriched in both the MMS- and Me-lex-resistant libraries. Expression of the L180F single mutant conferred 4.4-fold enhanced survival at the high dose of MMS used for selection. The homogeneous L180F mutant enzyme exhibited 2.2-fold reduced excision of 3-methyladenine and 7.3-fold reduced excision of 7-methylguanine from methylated calf thymus DNA. Decreased excision of methylated bases by the mutant glycosylase could promote survival at high MMS concentrations, where the capacity of downstream enzymes to process toxic BER intermediates may be saturated. The mutant also displayed 6.6- and 3.0-fold reduced excision of 1,N-6-ethenoadenine and hypoxanthine from oligonucleotide substrates, respectively, and a 1.7-fold increase in binding to abasic site-containing DNA. Our work provides in vivo evidence for the substrate binding mechanism deduced from crystal structures, illuminates the function of Leu(180) in wild-type human AAG, and is consistent with a role for balanced expression of BER enzymes in damage survival. (c) 2008 Elsevier B.V. All rights reserved.