Dissecting the broad substrate specificity of human 3-methyladenine-DNA glycosylase
Dissecting the broad substrate specificity of human 3-methyladenine-DNA glycosylase
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DOI：
10.1074/jbc.m312232200
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发表时间：
2004-03-12
影响因子：
4.8
通讯作者：
Ellenberger, T
中科院分区：
文献类型：
--
作者：
O'Brien, PJ;Ellenberger, T
人类烷基腺嘌呤-DNA糖基化酶（AAG）催化切除广泛的修饰碱基，保护基因组免受许多类型的烷基化和氧化DNA损伤。我们已经研究了AAG如何歧视正常的DNA碱基，同时容纳一组结构多样的病变的碱基，通过测量AAG催化的（k（st））和自发的N-糖苷键水解（k（非））的损坏和未损坏的DNA寡核苷酸的速率。不同碱基切除的速率增强表明，AAG最擅长切除脱氨基的损伤次黄嘌呤（k（st）/k（non）= 10（8）），表明酶活性可能是响应于该损伤而进化的。切除正常和修饰的嘌呤核碱基的速率增强的比较提供了证据，AAG通过与腺嘌呤和鸟嘌呤的环外氨基的空间冲突排除正常嘌呤。然而，甲基化嘌呤在化学上更不稳定，并且仅需要适度的速率增强来有效切除它们。碱基翻转也有助于特异性，因为不稳定的错配碱基对是比稳定的沃森-克里克对更好的底物，并且AAG识别的许多病变在其碱基配对能力方面受到损害。这些研究结果表明，AAG调和广泛的底物耐受性与生物学的必要性，以避免正常的DNA，从活性位点排除正常的碱基，而不是通过具体识别每个病变。
Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (k(st)) and spontaneous N-glycosidic bond hydrolysis (k(non)) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (k(st)/k(non) = 10(8)), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable Watson-Crick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion.