Mutagenesis and repair of butadiene DNA adducts

丁二烯 DNA 加合物的诱变和修复

• 批准号: 6340949
• 负责人: R. Stephen Lloyd
• 金额: $ 26.67万
• 依托单位: SOUTHERN UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6340949
• 关键词: DNA damage; DNA repair; adduct; animal genetic material tag; cooperative study; crosslink; diene; environmental toxicology; minority institution research support; mutagens; nucleic acid structure; transfection /expression vector

Although butadiene is a known mutagen and suspected carcinogen, the exact identity of the DNA adducts that are responsible for these events are not known. Furthermore, no studies have been conducted that ascertain the DNA repair mechanisms that are responsible for the removal of butadiene-modified nucleotides. In order to address these fundamental issues of butadiene-induced mutagenecity, as part of a prior collaboration with the Department of Chemistry at Vanderbilt University, oligonucleotides have been synthesized that contain site and stereospecific butadiene adducts: the R and S stereochemistries of the mono and diepoxides at N2-guanine and N6-adenine, as well as N6-N6 adenine and N2-N2-guanine intrastrand cross-links. The availability of these reagents facilitates the study of the molecular mechanisms of butadiene-induced mutagenesis and repair. This will be accomplished by engineering these modified DNAs into larger DNA templates that are suitable for detailed in vitro polymerization studies; these will include analyses of prokaryotic and eukaryotic replicative and repair polymerases. These same DNAs will be engineered into shuttle vectors that are designed for in vivo replication in either prokaryotic or eukaryotic systems. Mutagenic spectrum and frequencies will be assessed using differential hybridization strategies that are amenable to very high through-put of vectors. Additionally, collaborations between the Lloyd and Van Houten laboratories will establish which of the butadiene-modified DNAs are subject to correction by nucleotide excision repair using the E. coli UvrABC complex as the prototype. Fur collaborations are planned between the Lloyd/Van Houten groups with the Albrecht and Muganda groups, that are designed to measure rates of repair in cells exposed to butadiene metabolites using quantitative PCR assays. Finally, butadiene- induced DNA damage and repair will be measured in DNA repair proficient and deficient mice that have been exposed to butadiene gas; this study is in collaboration with Dr. Ward.

尽管丁二烯是一种已知的诱变剂和可疑的致癌物，但尚不清楚导致这些事件的DNA加合物的确切身份。此外，尚未进行研究，以确定负责去除丁二烯修饰的核苷酸的DNA修复机制。为了解决丁二烯引起的诱变的这些基本问题，作为与范德比尔特大学化学系的先前合作的一部分，已经合成了寡核苷酸，其中包含位点和立体的丁二烯加合物：R and diepoxiDes of Mono和DiapoxiDes conterepoxides在N2-瓜氨酸和N6-腺嘌呤，以及N6-N6腺嘌呤和N2-N2-瓜氨酸内氨链交联。这些试剂的可用性有助于研究丁二烯诱导的诱变和修复的分子机制。这将通过将这些改良的DNA设计成较大的DNA模板来实现，这些DNA适用于详细的体外聚合研究；这些将包括对原核生物和真核复制和修复聚合酶的分析。这些相同的DNA将被设计为用于原核或真核系统中体内复制的航天飞机载体。诱变光谱和频率将使用差分杂交策略进行评估，这些杂交策略可容纳非常高的载体。此外，劳埃德（Lloyd）和范·霍顿（Van Houten）实验室之间的合作将确定哪种丁二烯修饰的DNA可以通过使用大肠杆菌uvrabc复合物作为原型进行核苷酸切除修复进行校正。计划在劳埃德/范·霍顿（Lloyd/Van Houten）组与阿尔布雷希特（Albrecht）和穆加达（Muganda）组之间进行毛皮协作，这些群体旨在使用定量PCR测定法测量暴露于丁二烯代谢产物的细胞的维修率。最后，将在DNA修复中测量丁二烯诱导的DNA损伤和修复，熟练且缺乏暴露于丁二烯气体的小鼠。这项研究与沃德博士合作。