CHEMISTRY AND BIOLOGY OF MALONDIALDEHYDE DNA ADDUCTS

丙二醛 DNA 加合物的化学和生物学

• 批准号: 6514697
• 负责人: LAWRENCE J. MARNETT
• 金额: $ 38.04万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514697
• 关键词: DNA damage; DNA directed DNA polymerase; DNA replication; Escherichia coli; adduct; adenine analog; chemical addition; chemical kinetics; crosslink; enzyme activity; guanine analog; malonaldehyde; oligonucleotides; site directed mutagenesis; transfection /expression vector

Considerable attention has been focused recently on endogenous metabolic products as sources of DNA damage. Our laboratory has demonstrated that DNA adducts derived from the lipid oxidation product, malondialdehyde (MDA), are present in genomic DNA of healthy human beings. The major MDA-DNA adduct is a pyrimidopurinone that we call M1G; we and others have detected M1G in DNA from human liver, leukocytes, pancreas, and breast. Its identity in liver and leukocyte DNA has been verified by mass spectroscopy. Site-specific mutagenesis experiments with double-stranded viral genomes demonstrate that M1G induces mutations to T and to A in Escherichia coli and that it is repaired by nucleotide excision repair and by mismatch repair. Recently, we discovered that M1G undergoes reversible hydrolytic ring-opening to N2-oxopropenyl-G when it is present in duplex DNA opposite dC residues. This finding has important implications for the biological responses elicited by M1G, because it establishes that M1G is an electrophile in DNA with a reactive center located in the major groove. Its ring-opening product, N2-oxopropenyl-dG, presents an electrophilic center in the minor groove. Random mutagenesis experiments in bacterial and human cells suggest that other MDA-DNA adducts may be biologically important endogenous lesions, as well. Foremost among these are N6-oxopropenyl-A (M1A) and an enaminoimine N2-G-N2-G interstrand cross-link. We seek to define the chemistry of MDA-DNA adducts and to relate it to the biology that they exert. In the present application, we propose to 1) determine the kinetics of ring-opening and ring-closing of M1G and N2-oxopropenyl-G in different forms of DNA; 2) define the chemistry of of M1G-mediated DNA-DNA and DNA-protein cross-link formation; 3) determine the kinetics and products of bypass of M1G, N2- oxopropenyl-G, M1A, and an N2-G-N2-G trimethylene cross-link by the Klenow fragment of DNA polymerase I and by DNA polymerase V; and 4) determine the effects of M1G, N2-oxopropenyl-G, M1A, and an N2-G-N2-G trimethylene cross-link on DNA replication in bacterial and mammalian cells. The results of these experiments will define the chemistry and biology of a family of structurally dynamic lesions in the genome derived from an endogenous metabolic product of lipid oxidation.

内源性代谢产物是DNA损伤的来源，近年来引起了人们的广泛关注。 我们的实验室已经证明，来自脂质氧化产物丙二醛（MDA）的DNA加合物存在于健康人的基因组DNA中。 主要的MDA-DNA加合物是一种嘧啶嘌呤酮，我们称之为M1 G;我们和其他人已经在人类肝脏、白细胞、胰腺和乳腺的DNA中检测到了M1 G。其在肝脏和白细胞DNA中的身份已通过质谱法验证。 双链病毒基因组的位点特异性诱变实验表明，M1 G诱导大肠杆菌中的T和A突变，并通过核苷酸切除修复和错配修复进行修复。最近，我们发现，M1 G经历可逆的水解开环N2-oxopropyl-G时，它是存在于双链DNA相对dC残基。 这一发现对M1 G引起的生物反应具有重要意义，因为它确定了M1 G是DNA中的亲电体，其反应中心位于大沟中。 其开环产物N2-oxopropyl-dG在小沟中呈现亲电中心。 细菌和人类细胞中的随机诱变实验表明，其他MDA-DNA加合物也可能是生物学上重要的内源性病变。 其中最重要的是N6-氧代丙烯基-A（M1A）和烯氨基亚胺N2-G-N2-G链间交联。 我们试图定义MDA-DNA加合物的化学性质，并将其与它们发挥的生物学作用联系起来。在本申请中，我们提出1）确定M1 G和N2-氧代丙烯基-G在不同形式的DNA中的开环和闭环动力学; 2）定义M1 G介导的DNA-DNA和DNA-蛋白质交联形成的化学; 3）测定M1 G，N2-氧代丙烯基-G，M1A，以及通过DNA聚合酶I的Klenow片段和DNA聚合酶V的N2-G-N2-G三亚甲基交联;和4）确定M1 G、N2-氧代丙烯基-G、M1A和N2-G-N2-G三亚甲基交联对细菌和哺乳动物细胞中DNA复制的影响。 这些实验的结果将定义来自脂质氧化的内源性代谢产物的基因组中的结构动态病变家族的化学和生物学。