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).

该项目的目标是确定DNA修复和复制途径如何在系统变化的序列背景下响应DNA加合物8-氧鸟嘌呤（8- oxoG）的存在。热点地区受损鸟嘌呤的突变频率可能与它们的侧翼环境有关。为了解决这种可能性，将合成一系列包含8-oxoG的16个寡核苷酸，其两侧是所有可能的5‘和3’基变体。这些寡核苷酸将被放入病毒载体中，在转染大肠杆菌后测定体内突变频率。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结合的影响；而表达一种或两种糖酵解酶的细胞将决定哪个修复系统受序列背景的影响最大，这是由它们的相对突变频率决定的。这些寡核苷酸也将与它们的互补体杂交，形成短的双链，用于体外研究。将用热紫外光谱分析双相物，以确定与不含病变的对照物相比的自由能、焓和熔化温度。含有8-oxoG的双链也将被放射性标记，并用不同浓度的MutM或MutY消化，以确定酶-底物复合物的修复速率和解离常数。这些物理化学数据将与在每个序列背景下发现的突变频率相关联，以努力模拟选择性突变或氧化DNA损伤的修复（分别为热点和冷点）。