Lee Chun-Yue I, Delaney James C, Kartalou Maria, Lingaraju Gondichatnahalli M, Maor-Shoshani Ayelet, Essigmann John M, Samson Leona D
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2009 Mar 10;48(9):1850-61. doi: 10.1021/bi8018898.
The human 3-methyladenine DNA glycosylase (AAG) recognizes and excises a broad range of purines damaged by alkylation and oxidative damage, including 3-methyladenine, 7-methylguanine, hypoxanthine (Hx), and 1,N(6)-ethenoadenine (epsilonA). The crystal structures of AAG bound to epsilonA have provided insights into the structural basis for substrate recognition, base excision, and exclusion of normal purines and pyrimidines from its substrate recognition pocket. In this study, we explore the substrate specificity of full-length and truncated Delta80AAG on a library of oligonucleotides containing structurally diverse base modifications. Substrate binding and base excision kinetics of AAG with 13 damaged oligonucleotides were examined. We found that AAG bound to a wide variety of purine and pyrimidine lesions but excised only a few of them. Single-turnover excision kinetics showed that in addition to the well-known epsilonA and Hx substrates, 1-methylguanine (m1G) was also excised efficiently by AAG. Thus, along with epsilonA and ethanoadenine (EA), m1G is another substrate that is shared between AAG and the direct repair protein AlkB. In addition, we found that both the full-length and truncated AAG excised 1,N(2)-ethenoguanine (1,N(2)-epsilonG), albeit weakly, from duplex DNA. Uracil was excised from both single- and double-stranded DNA, but only by full-length AAG, indicating that the N-terminus of AAG may influence glycosylase activity for some substrates. Although AAG has been primarily shown to act on double-stranded DNA, AAG excised both epsilonA and Hx from single-stranded DNA, suggesting the possible significance of repair of these frequent lesions in single-stranded DNA transiently generated during replication and transcription.
人类3-甲基腺嘌呤DNA糖基化酶（AAG）能够识别并切除多种因烷基化和氧化损伤而受损的嘌呤，包括3-甲基腺嘌呤、7-甲基鸟嘌呤、次黄嘌呤（Hx）和1,N(6)-乙烯腺嘌呤（εA）。与εA结合的AAG晶体结构为底物识别、碱基切除以及正常嘌呤和嘧啶从其底物识别口袋中被排除的结构基础提供了见解。在本研究中，我们在一个包含结构多样碱基修饰的寡核苷酸文库上探究了全长和截短的Delta80AAG的底物特异性。检测了AAG与13种受损寡核苷酸的底物结合和碱基切除动力学。我们发现AAG能与多种嘌呤和嘧啶损伤结合，但仅切除其中少数几种。单轮切除动力学表明，除了众所周知的εA和Hx底物外，1-甲基鸟嘌呤（m1G）也能被AAG高效切除。因此，与εA和乙醇腺嘌呤（EA）一样，m1G是AAG和直接修复蛋白AlkB共有的另一种底物。此外，我们发现全长和截短的AAG都能从双链DNA中切除1,N(2)-乙烯鸟嘌呤（1,N(2)-εG），尽管效率较低。尿嘧啶能从单链和双链DNA中被切除，但仅能被全长AAG切除，这表明AAG的N端可能会影响某些底物的糖基化酶活性。尽管主要表明AAG作用于双链DNA，但AAG能从单链DNA中切除εA和Hx，这表明修复复制和转录过程中瞬时产生的单链DNA中这些常见损伤可能具有重要意义。
