FLANKING BASE SEQUENCE ON MUTAGENICITY OF 8 OXOGUANINE

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)在系统变化的序列环境中的存在。热点中受损鸟嘌呤的突变频率可能受其侧翼环境的支配。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，两侧有所有可能的5‘和3’基变体。这些寡核苷酸将被放入病毒载体中，用于体内突变频率的测定。缺乏已知的两种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误掺入的影响；而表达一种或两种糖酵解酶的细胞将根据它们的相对突变频率确定哪个修复系统受序列背景的影响最大。这些寡核苷酸还将与它们的补体杂交，形成短双链，用于体外研究。双链体将通过热紫外光谱进行分析，以确定与非损伤对照相比的自由能、熔化热焓和熔化温度。含有8-oxoG的双链也将被不同浓度的MutM或MutY标记和消化，以确定酶-底物复合体的修复速度和解离常数。这些物理化学数据将与每个序列背景下发现的突变频率相关联，以努力对氧化DNA损伤(分别是热点和冷点)的选择性突变或修复进行建模。

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).