EXOCYCLIC ADDUCTS--SITE-SPECIFIC MUTAGENESIS

外环加合物——位点特异性诱变

• 批准号: 6102495
• 负责人: Arthur Patrick Grollman
• 金额: $ 19.75万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6102495
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; N glycosidase; adduct; chemical carcinogenesis; chemical models; crosslink; crystallization; endonuclease; mutagens; nuclear magnetic resonance spectroscopy; nucleic acid metabolism; site directed mutagenesis; thermodynamics; tissue /cell culture; transfection /expression vector

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.

描述：（申请人的描述）该项目为本计划提供了生物学重点。 在其中，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 的结构和热力学特性联系起来。 我们的实验重点关注外环 DNA 加合物和氧化损伤、内源性 DNA 损伤形式，它们可能在人类癌症的发生中发挥重要作用。使用包含单个确定的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位点特异性病变的诱变特异性，并研究序列背景对跨病变合成过程中核苷酸错误掺入的影响。 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无碱基位点。在距损伤位点最多 5 个碱基的地方观察到核苷酸错误掺入率增加，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，将这些效应与受损 DNA 的热力学和结构特性联系起来。 我们还将研究校对对跨损伤综合的影响。 DNA损伤的准确修复对于生物体的生存至关重要。我们将通过确定与不可水解底物复合的选定 DNA 糖基化酶的晶体结构来研究 DNA 修复酶识别受损 DNA 的机制。 此外，我们希望分离和表征新型 DNA 糖基化酶，这些酶可以选择性地从 DNA 中切除环外加合物。