Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Am Chem Soc. 2009 Dec 16;131(49):17742-3. doi: 10.1021/ja908378y.
The duplex structure of DNA, with its internal base pairing, protects the nucleobases from chemical damage, but it also poses a barrier to DNA-modifying enzymes, including the enzymes that recognize and repair DNA damage. It is known that unpaired (or bulged) nucleotides are significantly more accessible, but it is not known whether they might be recognized by nucleotide-flipping enzymes. We have investigated this question with human alkyladenine DNA glycosylase (AAG). AAG recognizes a wide variety of structurally disparate lesions, including deoxyinosine (I), which results from the spontaneous oxidative deamination of adenosine, and catalyzes the hydrolysis of the N-glycosidic bond to release the lesion base and initiate the base excision repair pathway. We used single-turnover kinetics to characterize the reactions of AAG with synthetic 25-mer oligonucleotides containing a single I lesion in single-stranded, mismatched, or single-nucleotide bulge contexts. We found that AAG has the highest catalytic efficiency toward a lesion that is presented in a single-nucleotide bulge. In contrast, AAG has more than 2000-fold reduced catalytic efficiency toward a single-stranded I-containing oligonucleotide relative to the duplexes. We have observed 20-fold differences in catalytic efficiency for the excision of the presumed biological target (paired with T) relative to alternative pairings such as C that might be formed by the replication of an unrepaired I. Furthermore, a linear free-energy relationship shows a strong inverse correlation between duplex stability and catalytic efficiency (slope = -0.6 to -1.0), indicating that gaining access to the base lesion provides a substantial barrier to AAG-catalyzed initiation of DNA repair. The observation that AAG recognizes a single-nucleotide bulge as efficiently as a mismatch implies that the recognition of DNA damage is remarkably plastic.
DNA 的双螺旋结构及其内部碱基配对保护核碱基免受化学损伤，但它也为包括识别和修复 DNA 损伤的酶在内的 DNA 修饰酶构成了障碍。已知未配对（或膨出）核苷酸更容易接近，但尚不清楚它们是否可能被核苷酸翻转酶识别。我们用人烷基腺嘌呤 DNA 糖基化酶（AAG）研究了这个问题。AAG 识别多种结构不同的损伤，包括脱氧肌苷（I），它是由腺嘌呤自发氧化脱氨产生的，并催化 N-糖苷键的水解，释放损伤碱基并启动碱基切除修复途径。我们使用单次周转动力学来描述 AAG 与含有单链、错配或单核苷酸膨出的合成 25 聚核苷酸反应。我们发现 AAG 对单核苷酸膨出中呈现的损伤具有最高的催化效率。相比之下，相对于双链体，AAG 对含有单链 I 的寡核苷酸的催化效率降低了 2000 多倍。我们观察到，相对于可能通过未修复 I 的复制形成的替代配对（例如 C），从假定的生物靶标（与 T 配对）中切除的催化效率差异了 20 倍。此外，线性自由能关系显示双链体稳定性和催化效率之间存在很强的负相关（斜率=-0.6 至-1.0），表明获得碱基损伤的能力为 AAG 催化的 DNA 修复启动提供了实质性障碍。AAG 能够像错配一样有效地识别单核苷酸膨出的观察结果表明，DNA 损伤的识别具有显著的可塑性。
