Elucidating Mechanisms of Recognition and Excision of Damaged Bases by NEIL glycosylases

氧化应激通过修改DNA碱基来侵蚀DNA的完整性，并与癌症有关， 神经紊乱和衰老。Neil糖基酶启动氧化碱基损伤的碱基切除修复 通过催化N-糖苷键断裂到2‘-脱氧核糖，并能够去除 广泛的DNA碱基修饰。尼尔糖基酶具有独特的作用于多种不同的 双链DNA以外的环境，如单链DNA和G-四链，这表明这些酶 在修复、复制和转录中发挥核心作用。我们已经证明了海因损伤，即 在高氧化应激条件下形成，并在富含G的序列中形成，如G-四链，是 尼尔糖基酶的最佳底物。我们的实验室也是第一个在 RNA编辑和DNA修复通过显示由NEIL1进行的损伤处理受RNA编辑反应调节 这改变了酶的病变识别环中氨基酸位置242的密码子，切换到 从基因编码的赖氨酸到精氨酸的残基。这一变化改变了NEIL1糖基酶的比率 损伤和DNA上下文依赖的方式中的常量。病变身份和背景也会影响 NEIL1碱基切除的程度，这种损伤结合特性表明在调节复制和 抄写。我们还发现了NEIL1和NEIL3在移除 来自不同G-四链序列的海因损伤。不同G-四链序列的存在 在基因启动子中，奇怪的是在DNA修复酶本身中，表明观察到的差异 病变切除的程度和非生产性结合的存在可能会改变转录，以响应 氧化应激。这些观察结果进一步表明，Neil酶在经典误码率之外的过程中也有影响 进一步强调核酸交易之间紧密的相互依存关系。该项目将需要使用多个 涉及酶学、核酸化学、生物物理方法和细胞分析的多方面方法 探索NEIL1、2和3的损伤和上下文相关特性，以建立两者之间的直接联系 基于防止DNA突变的损伤识别和碱基切除的特定方面的分子缺陷 以及改变基因转录。

Oxidative stress erodes the integrity of DNA by modifying DNA bases and has been linked to cancer, neurological disorders and aging. The NEIL glycosylases initiate base excision repair of oxidized base lesions by catalyzing the cleavage of the N-glycosidic linkage to the 2’-deoxyribose and are capable of removing a wide array of modified DNA bases. The NEIL glycosylases are unique in acting in a variety of different contexts beyond duplex DNA, such as ssDNA, and G-quadruplexes, that has suggested that these enzymes play central roles in repair, replication and transcription. We have shown that hydantoin lesions, that are formed formed under conditions of high oxidative stress, and in G-rich sequences such as G-quadruplexes, are the best substrates for the NEIL glycosylases. Our laboratory was also the first to provide a direct link between RNA editing and DNA repair by showing lesion processing by NEIL1 is modulated by an RNA editing reaction that changes the codon for amino acid position 242 in the lesion recognition loop of the enzyme, switching the residue from the genomically encoded lysine to an arginine. This change alters NEIL1 glycosylase rate constants in a lesion and DNA context dependent manner. Lesion identity and context also influences the extent of NEIL1 base excision, and this lesion binding property suggest roles in regulating replication and transcription. We have also uncovered unique differences between the NEIL1 and NEIL3 in the removal of hydantoin lesions from different G-quadruplex sequences. The presence of different G-quadruplex sequences in gene promoters, curiously in the DNA repair enzymes themselves, suggests that the observed differences in extents of lesion excision, and the presence of non-productive binding, may alter transcription in response to oxidative stress. These observations further implicate NEIL enzymes in processes beyond classic BER, and further underscore the tight interdependence of nucleic acid transactions. This project will entail using a multi- faceted approach involving enzymology, nucleic acid chemistry, biophysical methods and cellular assays to probe the lesion and context dependent properties of NEIL1, 2 and 3 to make direct connections between molecular defects in particular aspects of damage recognition and base excision on preventing DNA mutations and altering gene transcription.