EXOCYCLIC ADDUCTS--SITE-SPECIFIC MUTAGENESIS

描述：(申请者描述)这个项目为这个项目提供了一个生物学的焦点。在它中，我们探索了涉及突变和DNA修复的分子和细胞事件，将我们的发现与受损DNA的结构和热力学性质联系起来。我们的实验重点放在外环DNA加合物和氧化损伤上，这是一种内源性的DNA损伤形式，可能在人类癌症的发生中发挥重要作用。使用包含单一定义的DNA加合物的新型双链穿梭载体系统，我们建议在人类细胞中建立定点定位损伤的突变特异性，并研究序列背景对跨损伤合成过程中核苷酸误掺入的影响。我们还将探索复杂DNA损伤的修复和致突变性，包括链间交联和双链基本位点。在距离损伤部位五个碱基的地方观察到核苷酸误掺入率增加，这表明这些事件发生在DNA的热力学不稳定区域。使用纯化的DNA聚合酶，我们计划在远离病变部位的位置建立DNA合成的效率和保真度，将这些影响与受损DNA的热力学和结构性质联系起来。我们还将调查校对对跨病变合成的影响。DNA损伤的准确修复对生物的生存至关重要。我们将通过确定与非水解性底物络合的选定DNA糖基酶的晶体结构来研究DNA修复酶识别受损DNA的机制。此外，我们希望分离和鉴定新的DNA糖基酶，这种酶可以选择性地从DNA中去除外环加合物。

DESCRIPTION: (Applicant's Description) This project provides a biological focus for this program. In it, we explore molecular and cellular events involved in mutagenesis and DNA repair, relating our findings to the structure and thermodynamic properties of damaged DNA. Our experiments focus on exocyclic DNA adducts and oxidative lesions, endogenous forms of DNA damage that may play an important role in the initiation of human cancer. Using a novel double strand shuttle vector system containing a single defined DNA adduct, we propose to establish in human cells the mutagenic specificity of site-specifically placed lesions and to investigate the effects of sequence context on nucleotide misincorporation during translesion synthesis. We also will explore the repair and mutagenicity of complex DNA damages, including interstrand crosslinks and bistrand abasic sites. Increased rates of nucleotide misincorporation have been observed up to five bases away from the site of damage, suggesting these events take place in a thermodynamically destabilized region of DNA. Using purified DNA polymerases, we plan to establish efficiency and fidelity of DNA synthesis at positions remote from the lesion site, relating these effects to thermodynamic and structural properties of damaged DNA. We also will investigate the effects of proofreading on translesion synthesis. Accurate repair of DNA damage is critical to the survival of living organisms. We will examine mechanisms by which DNA repair enzymes recognize damaged DNA by determining the crystal structures of selected DNA glycosylases complexed to non-hydrolyzable substrates. In addition, we hope to isolate and characterize novel DNA glycosylases that selectively excise exocyclic adducts from DNA.