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

氧化应激通过修饰DNA碱基侵蚀DNA的完整性，并与癌症有关， 神经系统疾病和衰老。NEIL糖基化酶启动氧化基底损伤的基底切除修复 通过催化N-糖苷键裂解至2 ′-脱氧核糖，并且能够除去 大量的修饰DNA碱基NEIL糖基化酶是独特的，在多种不同的酶中起作用。 双链体DNA之外的背景，如ssDNA和G-四链体，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，乙内酰脲损伤， 在高氧化应激条件下形成的，以及在富含G的序列如G-四链体中， NEIL糖基化酶的最佳底物。我们的实验室也是第一个提供直接联系， RNA编辑和DNA修复通过显示NEIL 1的损伤处理受到RNA编辑反应的调节 改变酶的损伤识别环中氨基酸位置242的密码子， 从基因组编码的赖氨酸残基到精氨酸。这种变化改变NEIL 1糖基化酶速率 在损伤和DNA上下文依赖的方式常数。病变的性质和背景也会影响 NEIL 1碱基切除的程度，这种损伤结合特性表明在调节复制和 转录。我们还发现了NEIL 1和NEIL 3在去除 不同G-四链体序列的乙内酰脲损伤。不同G-四链体序列的存在 在基因启动子中，奇怪的是在DNA修复酶本身中，这表明在基因启动子中观察到的差异， 损伤切除的程度和非生产性结合的存在，可能会改变转录， 氧化应激这些观察结果进一步暗示了NEIL酶在经典BER之外的过程中， 进一步强调了核酸交易的紧密相互依赖性。该项目将需要使用多- 涉及酶学、核酸化学、生物物理方法和细胞测定的多方面方法， 探索NEIL 1、2和3的病变和上下文相关属性，以建立直接联系 损伤识别和碱基切除等特定方面的分子缺陷对防止DNA突变的作用 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.