Base excision repair (BER) enzymes are genomic superheroes that stealthily and accurately identify and remove chemically modified DNA bases. DNA base modifications erode the informational content of DNA and underlie many disease phenotypes, most conspicuously, cancer. The “OG” of oxidative base damage, 8-oxo-7,8-dihydroguanine (OG), is particularly insidious due to its miscoding ability that leads to the formation of rare, pro-mutagenic OG:A mismatches. Thwarting mutagenesis relies on the capture of OG:A mismatches prior to DNA replication and removal of the mis-inserted adenine by MutY glycosylases to initiate BER. The threat of OG and the importance of its repair are underscored by the association between inherited dysfunctional variants of the MutY human homologue (MUTYH) and colorectal cancer, known as MUTYH-associated polyposis (MAP). Our functional studies of the two founder MUTYH variants revealed that both have compromised activity and a reduced affinity for OG:A mismatches. Indeed, these studies underscored the challenge of the recognition of OG:A mismatches that are only subtly structurally different than T:A base pairs. Since the original discovery of MAP, many MUTYH variants have been reported, with most considered to be “variants of uncertain significance.” To reveal features associated with damage recognition and adenine excision by MutY and MUTYH, we have developed a multipronged chemical biology approach combining enzyme kinetics, X-ray crystallography, single-molecule visualization, and cellular repair assays. In this review, we highlight recent work in our laboratory where we defined MutY structure–activity relationship (SAR) studies using synthetic analogs of OG and A in cellular and in vitro assays. Our studies revealed the 2-amino group of OG as the key distinguishing feature of OG:A mismatches. Indeed, the unique position of the 2-amino group in the major groove of OG_syn:A_anti mismatches provides a means for its rapid detection among a large excess of highly abundant and structurally similar canonical base pairs. Furthermore, site-directed mutagenesis and structural analysis showed that a conserved C-terminal domain β-hairpin “FSH’’ loop is critical for OG recognition with the “His” serving as the lesion detector. Notably, MUTYH variants located within and near the FSH loop have been associated with different forms of cancer. Uncovering the role(s) of this loop in lesion recognition provided a detailed understanding of the search and repair process of MutY. Such insights are also useful to identify mutational hotspots and pathogenic variants, which may improve the ability of physicians to diagnose the likelihood of disease onset and prognosis. The critical importance of the “FSH” loop in lesion detection suggests that it may serve as a unique locus for targeting probes or inhibitors of MutY/MUTYH to provide new chemical biology tools and avenues for therapeutic development.

中文翻译：

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FSHing 用于 DNA 损伤：8-氧代鸟嘌呤：腺嘌呤错配的 MutY 检测的关键特征

碱基切除修复 (BER) 酶是基因组超级英雄，可以秘密且准确地识别和去除化学修饰的 DNA 碱基。 DNA 碱基修饰会侵蚀 DNA 的信息内容，并成为许多疾病表型的基础，其中最引人注目的是癌症。氧化碱基损伤的“OG”8-oxo-7,8-二氢鸟嘌呤 (OG) 特别隐蔽，因为它的错误编码能力会导致罕见的促突变 OG:A 错配的形成。阻止诱变依赖于在 DNA 复制之前捕获 OG:A 错配，并通过 MutY 糖基化酶去除错误插入的腺嘌呤以启动 BER。 MutY 人类同源物 (MUTYH) 的遗传性功能障碍变异与结直肠癌（称为 MUTYH 相关息肉病 (MAP)）之间的关联强调了 OG 的威胁及其修复的重要性。我们对两个创始人 MUTYH 变体的功能研究表明，两者的活性均受到损害，并且对 OG:A 错配的亲和力降低。事实上，这些研究强调了识别 OG:A 错配的挑战，这些错配仅在结构上与 T:A 碱基对略有不同。自 MAP 最初发现以来，已报道了许多 MUTYH 变体，其中大多数被认为是“意义不确定的变体”。为了揭示与 MutY 和 MUTYH 损伤识别和腺嘌呤切除相关的特征，我们开发了一种多管齐下的化学生物学方法，结合了酶动力学、X 射线晶体学、单分子可视化和细胞修复测定。在这篇综述中，我们重点介绍了我们实验室最近的工作，其中我们在细胞和体外测定中使用 OG 和 A 的合成类似物定义了 MutY 结构-活性关系 (SAR) 研究。我们的研究揭示了 OG 的 2-氨基是 OG:A 错配的关键区别特征。事实上，OG _syn :A _anti的大沟中 2-氨基的独特位置错配提供了一种在大量高丰度且结构相似的规范碱基对中快速检测的方法。此外，定点诱变和结构分析表明，保守的C端结构域β-发夹“FSH”环对于以“His”作为病变检测器的OG识别至关重要。值得注意的是，位于 FSH 环内部和附近的 MUTYH 变异与不同形式的癌症有关。揭示该循环在病变识别中的作用可以帮助人们详细了解 MutY 的搜索和修复过程。这些见解对于识别突变热点和致病变异也很有用，这可能会提高医生诊断疾病发病可能性和预后的能力。 “FSH”环在病变检测中的至关重要性表明，它可以作为 MutY/MUTYH 靶向探针或抑制剂的独特位点，为治疗开发提供新的化学生物学工具和途径。