Conlon Savannah G, Khuu Cindy, Trasviña-Arenas Carlos H, Xia Tian, Hamm Michelle L, Raetz Alan G, David Sheila S
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States.
Graduate Program in Chemistry and Chemical Biology, University of California, Davis, One Shields Avenue, Davis, California 95616, United States.
ACS Cent Sci. 2024 Jan 26;10(2):291-301. doi: 10.1021/acscentsci.3c00784. eCollection 2024 Feb 28.
The base excision repair glycosylase MUTYH prevents mutations associated with the oxidatively damaged base, 8-oxo-7,8-dihydroguanine (OG), by removing misincorporated adenines from OG:A mispairs. Defects in OG:A repair in individuals with inherited MUTYH variants are correlated with the colorectal cancer predisposition syndrome known as -associated polyposis (MAP). Herein, we reveal key structural features of OG required for efficient repair by human MUTYH using structure-activity relationships (SAR). We developed a GFP-based plasmid reporter assay to define SAR with synthetically generated OG analogs in human cell lines. Cellular repair results were compared with kinetic parameters measured by adenine glycosylase assays . Our results show substrates lacking the 2-amino group of OG, such as 8OI:A (8OI = 8-oxoinosine), are not repaired in cells, despite being excellent substrates in adenine glycosylase assays, new evidence that the search and detection steps are critical factors in cellular MUTYH repair functionality. Surprisingly, modification of the O8/N7H of OG, which is the distinguishing feature of OG relative to G, was tolerated in both MUTYH-mediated cellular repair and adenine glycosylase activity. The lack of sensitivity to alterations at the O8/N7H in the SAR of MUTYH substrates is distinct from previous work with bacterial MutY, indicating that the human enzyme is much less stringent in its lesion verification. Our results imply that the human protein relies almost exclusively on detection of the unique major groove position of the 2-amino group of OG within OG:A mispairs to select contextually incorrect adenines for excision and thereby thwart mutagenesis. These results predict that MUTYH variants that exhibit deficiencies in OG:A detection will be severely compromised in a cellular setting. Moreover, the reliance of MUTYH on the interaction with the OG 2-amino group suggests that disrupting this interaction with small molecules may provide a strategy to develop potent and selective MUTYH inhibitors.
碱基切除修复糖基化酶MUTYH通过从OG:A错配中去除错掺入的腺嘌呤，防止与氧化损伤碱基8-氧代-7,8-二氢鸟嘌呤（OG）相关的突变。携带遗传性MUTYH变体的个体中OG:A修复缺陷与称为MUTYH相关息肉病（MAP）的结直肠癌易感性综合征相关。在此，我们利用构效关系（SAR）揭示了人MUTYH有效修复所需的OG关键结构特征。我们开发了一种基于绿色荧光蛋白的质粒报告基因检测方法，以确定人细胞系中与合成生成的OG类似物的SAR。将细胞修复结果与腺嘌呤糖基化酶检测测得的动力学参数进行比较。我们的结果表明，缺乏OG 2-氨基的底物，如8OI:A（8OI = 8-氧代肌苷），在细胞中无法修复，尽管它们在腺嘌呤糖基化酶检测中是优秀的底物，这一新证据表明搜索和检测步骤是细胞MUTYH修复功能的关键因素。令人惊讶的是，OG的O8/N7H修饰（这是OG相对于G的显著特征）在MUTYH介导的细胞修复和腺嘌呤糖基化酶活性中均被耐受。MUTYH底物的SAR对O8/N7H改变缺乏敏感性与之前关于细菌MutY的研究不同，表明人酶在损伤验证方面的严格程度要低得多。我们的结果意味着人蛋白质几乎完全依赖于检测OG:A错配中OG 2-氨基在独特大沟位置来选择上下文错误的腺嘌呤进行切除，从而防止诱变。这些结果预测，在细胞环境中，表现出OG:A检测缺陷的MUTYH变体将受到严重损害。此外，MUTYH对与OG 2-氨基相互作用的依赖表明，用小分子破坏这种相互作用可能提供一种开发强效和选择性MUTYH抑制剂的策略。
