Repair of Oxidized Bases in Mammalian Genomes

描述（由申请人提供）：活性氧（ROS），内源性作为呼吸副产物产生，外源性由辐射和其他基因毒素产生，可诱导细胞基因组中的各种碱基损伤和DNA链断裂，这些损伤大多通过碱基切除修复（BER）途径修复，由DNA糖基化酶切除碱基损伤启动。在缺乏OGG1和NTH1这两种主要的哺乳动物糖基酶的小鼠中，氧化碱基损伤的积累没有很强的表型，这表明额外的糖基酶可以修复活跃的基因组序列。在我们之前的项目期间，我们表征了人类糖基酶（NEIL1和NEIL2）具有重叠的底物偏好，如OGG1和NTH1。然而，只有NEILs从单链或气泡dna中切除病变。NEIL2与RNA聚合酶II、NEIL1与滑动钳PCNA和其他DNA复制相关蛋白的稳定相互作用使我们假设NEIL1和-2分别在复制（RAR）或转录相关修复中具有优先作用。我们进一步假设NEIL1优先修复前导或滞后模板链中的碱基损伤（复制修复），并去除新生DNA链中合并的碱基损伤（复制后修复）。在辐射和氧化应激的反应中，多蛋白复合物的形成也可能受到组分蛋白的共价修饰的影响，这使我们假设，通过利用不同的DNA聚合酶和其他蛋白质，包括RPA、FEN1（由PCNA激活）和具有DNA解螺旋酶活性的Werner或Bloom蛋白，不同的修复复合物被组装起来修复内源性与辐射/ ros诱导的损伤，以及G1期与s期细胞。在这个竞争性更新项目中，我们将通过测试是否(1)NEIL1在G1期和s期细胞中形成不同的修复复合物，并对氧化应激作出反应，来检验这一假设的各个方面；(2) NEIL1通过与复制复合体相互作用，在复制过程中优先修复模板DNA和新生DNA中的碱基损伤；(3)相对于非复制质粒，复制质粒的损伤在体内更容易修复；(4) neil1诱导的复制DNA链损伤位点的切割导致双链断裂。健康相关性：这些研究的成功完成不仅将建立RAR的新概念，而且将阐明修复蛋白不同复合物之间的各种相互作用。这种对修复复合体相互作用界面的基本理解可以用于开发化疗/放疗期间致敏肿瘤细胞或保护健康细胞的治疗干预策略。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS), endogenously generated as respiration by-products and exogenously by radiation and other genotoxins, induce a variety of base damage and DNA strand breaks in cell genomes, which are mostly repaired via the base excision repair (BER) pathway, initiated with excision of base lesions by DNA glycosylases. Accumulation of oxidized base damage without a strong phenotype in mice lacking OGG1 and NTH1, the major mammalian glycosylases, suggested that additional glycosylases repair active genomic sequences. In our previous project period we characterized human glycosylases (NEIL1 and NEIL2) with overlapping substrate preferences as OGG1 and NTH1. However, only the NEILs excise lesions from single-stranded or bubble DNAs. Stable interaction of NEIL2 with RNA polymerase II, and of NEIL1 with the sliding clamp PCNA, and other DNA replication-associated proteins led us to hypothesize a preferential role for NEIL1 and -2 in replication (RAR)- or transcription-associated repair, respectively. We further postulate that NEIL1 preferentially repairs base damage in leading or lagging template strand (preplicative repair), and also removes incorporated base lesion in the nascent DNA strand (post-replicative repair). Formation of multiprotein complexes in response to radiation and oxidative stress, which may also be affected by covalent modification of component proteins, led us to hypothesize that distinct repair complexes are assembled for repairing endogenous vs. radiation/ROS-induced damage, and in G1 vs. S-phase cells, by utilizing distinct DNA polymerases and other proteins including RPA, FEN1 (activated by PCNA) and Werner or Bloom protein with DNA helicase activity. In this competing renewal project we will examine various facets of this hypothesis by testing whether (1) NEIL1 forms distinct repair complexes in G1 vs. S-phase cells, and in response to oxidative stress; (2) NEIL1 preferentially repairs base damage in both template and nascent DNA during replication by interacting with the replicating complex; (3) damage in replicating plasmid is preferentially repaired in vivo relative to nonreplicating plasmid; and (4) NEIL1-induced cleavage at the damage site of the replicating DNA strand leads to double-strand breaks. Health Relevance: Successful completion of these studies will not only establish the novel concept of RAR, but will also illuminate various interactions among repair proteins distinct complexes. Such basic understanding of interaction interfaces in repair complexes could be exploited in developing therapeutic intervention strategies for sensitizing tumor cells or protecting healthy cells during chemo/radiation therapy.