CHEMISTRY AND BIOLOGY OF MALONDIALDEHYDE DNA ADDUCTS

最近，作为DNA损伤来源的内源性代谢产物受到了相当大的关注。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。主要的丙二醛-DNA加合物是一种我们称为M1G的嘧啶并嘌呤酮；我们和其他人在人类肝脏、白细胞、胰腺和乳房的DNA中检测到了M1G。其在肝脏和白细胞DNA中的同源性已被质谱学证实。用双链病毒基因组进行的定点突变实验表明，M1G在大肠杆菌中诱导T和A突变，并通过核苷酸切除修复和错配修复来修复。最近，我们发现，当M1G存在于与DC残基相反的双链DNA中时，它会发生可逆的水解环开环反应，生成N-氧代丙烯-G。这一发现对M1G引起的生物反应具有重要的意义，因为它确立了M1G是DNA中的一个电泳体，其反应中心位于主槽中。其开环产物N_2-氧代丙烯基-DG在小凹槽中呈现亲电中心。在细菌和人类细胞中的随机突变实验表明，其他MDA-DNA加合物也可能是生物学上重要的内源性损伤。其中最重要的是N6-氧代丙烯-A(M1A)和氨基亚胺N_2-G-N_2-G链间交联物。我们试图定义丙二醛-DNA加合物的化学，并将其与它们发挥作用的生物学联系起来。在本应用中，我们建议1)测定不同形式DNA中M1G和N_2-氧代丙烯-G的开环和闭环动力学；2)确定M_1G介导的DNA-DNA和DNA-蛋白质交联形成的化学；3)测定DNA聚合酶I的Klenow片段和DNA聚合酶V对M_1G、N_2-氧代丙烯-G、M_1a和N_2-G-N_2-G三亚甲基交联物的旁路动力学和产物；以及4)确定M1G、N_2-氧代丙烯-G、M_1A和N_2-G-N_2-G三亚甲基交联物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基因组中一系列结构动态损伤的化学和生物学，这些损伤来自于脂质氧化的内源性代谢产物。

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.