FLANKING BASE SEQUENCE ON MUTAGENICITY OF 8 OXOGUANINE

8 氧鸟嘌呤致突变性的侧翼碱基序列

• 批准号: 6362773
• 负责人: Paul Thomas Henderson
• 金额: $ 4.02万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6362773
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; N glycosidase; NAD nucleosidase; adduct; chemical association; enzyme activity; enzyme substrate complex; gene mutation; guanine analog; nucleic acid hybridization; nucleic acid sequence; oligonucleotides; transfection; transfection /expression vector; ultraviolet spectrometry

DESCRIPTION The goal of this project is to determine how DNA repair and replication pathways respond to the presence of the DNA adduct 8-oxoguanine (8- oxoG) in a systematically varied sequence context. The mutation frequency of damaged guanine in hotspots is likely to be governed by their flanking context.. To address this possibility, a series of sixteen oligonucleotides containing 8-oxoG flanked by all possible 5'-and 3'- based variations will be synthesized. The oligonucleotides will be placed into viral vectors for in vivo determination of mutational frequency after transfection into E. coli. Cells lacking expression of the two known 8- oxoG base excision repair enzymes, MutM and MutY, will address the impact of sequence effects on polymerase misincorporation; while cells expressing either one or both glycolyases will determine which repair system is most affected by sequence context, as determined by their relative mutation frequencies. The oligonucleotides will also be hybridized to their complements to form short duplexes for in vitro studies. The duplexes will be analyzed by thermal UV spectroscopy to determine the free energy, enthalpy and temperature of melting compared to non-lesion containing controls. The 8-oxoG containing duplexes will also be radiolabeled and digested with MutM or MutY at various concentrations to determine the rate of repair and the dissociation constant of the enzyme-substrate complex. These physiochemical data will be correlated to the mutation frequency found for each sequence context in an effort to model selective mutation or repair of oxidative DNA lesions (hot- and coldspots-respectively).

描述本项目的目标是确定DNA修复和复制途径如何响应DNA加合物8-oxoguanine（8- oxoG）在系统变化的序列背景下的存在。热点中受损鸟嘌呤的突变频率可能受其侧翼背景的影响。为了解决这种可能性，将合成一系列十六种寡核苷酸，其含有侧翼为所有可能的基于5 '和3'的变异的8-oxoG。将寡核苷酸置于病毒载体中，用于在转染到E.杆菌缺乏两种已知的8- oxoG碱基切除修复酶MutM和MutY表达的细胞将解决序列效应对聚合酶错误掺入的影响;而表达一种或两种糖酵解酶的细胞将确定哪种修复系统最受序列背景的影响，如由其相对突变频率所确定的。寡核苷酸还将与其互补物杂交以形成用于体外研究的短双链体。将通过热UV光谱法分析双链体，以确定与不含病变的对照相比的自由能、焓和熔解温度。还将放射性标记含有8-oxoG的双链体，并用各种浓度的MutM或MutY消化，以确定修复速率和酶-底物复合物的解离常数。这些理化数据将与每个序列背景中发现的突变频率相关，以模拟氧化DNA损伤的选择性突变或修复（分别为热点和冷点）。