Mechanistic insight into the recognition of single-stranded and double-stranded DNA substrates by ABH2 and ABH3
Mechanistic insight into the recognition of single-stranded and double-stranded DNA substrates by ABH2 and ABH3
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DOI：
10.1039/c005148a
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发表时间：
2010-01-01
影响因子：
--
通讯作者：
Yang, Cai-Guang
中科院分区：
文献类型：
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作者：
Chen, Baoen;Liu, Hongchuan;Yang, Cai-Guang
人ABH 2和ABH 3蛋白在N-1-甲基腺嘌呤（1-meA）和N-3-甲基胞嘧啶（3-meC）核苷酸碱基的氧化去甲基化方面功能互补。ABH 3在体外与单链DNA（ssDNA）表现出更高的活性，而ABH 2在哺乳动物中作为主要的管家酶，用于有效地修复双链DNA（dsDNA）中内源性形成的烷基化损伤。在结构上，它们的整体蛋白质折叠是非常相似的，但最显着的差异发生在核苷酸识别盖和β-发夹基序。我们在这里提出了一个定点突变分析和基序交换的研究，以获得机制的洞察ABH 2和ABH 3的DNA底物选择。V101 A-F102 A双突变体显著降低了双链DNA中ABH 2的活性，表明该疏水区域似乎对损伤搜索和修复很重要。发现苯丙氨酸指F102对于ssDNA选择和修复也是至关重要的;然而，V101仅对ssDNA而非dsDNA显示出降低的去甲基化活性。ABH 2 R110 A突变体完全丧失甲基碱基修复活性，表明R110可能参与碱基翻转过程。E175和F124有助于核苷酸碱基特异性选择和修复活性位点的稳定化。此外，将ABH 3中的RED残基交换为ABH 2中的等效VFG残基赋予ABH 3与野生型ABH 2一样有效的dsDNA修复活性。令人惊讶的是，通过改变几个残基，ABH 3蛋白可以对ssDNA或dsDNA具有非常不同的选择性。该结果表明RED基序最有可能阻止ABH 3结合和dsDNA的修复。一致地，交换ABH 3与dsDNA的交联非常好，证实了这些残基在初始DNA链识别中的决定性作用。总体而言，这项工作提供了ABH 2和ABH 3的ssDNA和dsDNA偏好的结构特征的详细了解。
The human ABH2 and ABH3 proteins are functionally complementary in the oxidative demethylation of N-1-methyl adenine (1-meA) and N-3-methyl cytosine (3-meC) nucleotide bases. ABH3 displays higher activities with single-stranded DNA (ssDNA) in vitro, whereas ABH2 acts as the primary housekeeping enzyme in mammals for effectively repairing endogenously formed alkylated lesions in double-stranded DNA (dsDNA). Structurally, their overall protein folding is quite similar, but the most significant differences occur in the nucleotide recognition lid and the beta-hairpin motif. We present here a site-directed mutational analysis and motif-swapping study to gain mechanistic insight into DNA substrate selection by ABH2 and ABH3. A V101A-F102A double mutant notably reduced ABH2 activity in dsDNA, indicating that this hydrophobic region appears to be important for damage searching and repair. The phenylalanine finger F102 is found to be crucial for ssDNA selection and repair as well; however, V101 shows reduced demethylating activity for only ssDNA and not dsDNA. The ABH2 R110A mutant completely loses the methyl base repair activity, suggesting that R110 is likely to be involved in the base flipping process. E175 and F124 contribute to nucleotide base specific selection and stabilization in the active site for repair. Additionally, swapping the RED residues in ABH3 to equivalent VFG residues in ABH2 endows ABH3 with activity in dsDNA repair as efficient as wild-type ABH2. Surprisingly, by changing just a few residues, the ABH3 protein can have very different selectivity towards ssDNA or dsDNA. This result indicates that the RED motif most likely prevents ABH3 binding and repair of dsDNA. Consistently, swapped ABH3 cross-links with dsDNA very well, confirming the determining roles of these residues in the initial DNA strand recognition. Overall, this work has provided a detailed understanding of the structural features of the ssDNA and dsDNA preferences of ABH2 and ABH3.