Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-5606, United States.
Biochemistry. 2011 Mar 22;50(11):1864-74. doi: 10.1021/bi101856a. Epub 2011 Feb 3.
Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (εA), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs.
人类烷基腺嘌呤 DNA 糖基化酶 (AAG) 从 DNA 中定位并切除各种结构不同的烷基化和氧化嘌呤损伤，从而启动碱基切除修复途径。碱基损伤的识别需要将受损核苷酸翻转到两个保守的酪氨酸残基（Y127 和 Y159）之间相对开放的活性位点口袋中。我们已经将这些氨基酸突变为色氨酸，并测量了核苷酸翻转和碱基切除步骤的动力学影响。Y127W 和 Y159W 突变蛋白对含有 1,N(6)-乙撑腺嘌呤 (εA) 的 DNA 具有强大的糖苷酶活性，与野生型酶相比，其活性提高了 4 倍，这表明色氨酸荧光可能用于探测 DNA 结合和核苷酸翻转步骤。停流荧光用于比较色氨酸荧光和 εA 荧光随时间的变化。对于这两种突变体，色氨酸荧光均表现出两步结合，其速率常数与 εA 荧光变化观察到的基本相同。这些结果表明，AAG 形成了一个初始识别复合物，其中活性位点口袋受到干扰，受损碱基的堆积被破坏。完全核苷酸翻转后，色氨酸荧光进一步猝灭，同时 εA 荧光猝灭。尽管这些突变对 εA 的切除速率常数没有很大影响，但核苷酸翻转的速率常数却有很大影响，导致翻转平衡相对于野生型降低了 40-100 倍。这种影响主要归因于翻转的非翻转速率增加，但令人惊讶的是，Y159W 突变导致翻转速率常数增加了 5 倍。核苷酸翻转平衡的巨大影响解释了这些突变对更稳定碱基对中碱基损伤的糖苷酶活性产生更大的有害影响。
