Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic M

多环芳烃（PAHs）如苯并[a]芘（BaP）和苯并[c]菲（BcPh）是广泛存在的环境污染物，其中许多是哺乳动物的强致癌物。这些碳氢化合物通过细胞色素P450和环氧化物水解酶的作用进行代谢活化，生成高活性的湾区二醇环氧化物（DEs），主要在嘌呤碱基的外环氨基上形成共价DNA加合物。DNA聚合酶对修饰残基的错误复制导致突变，这可能是诱导癌症的第一步。16个海湾地区的去嘌呤加合物对一个给定的母体烃是代谢可能的。含有这些加合物的双寡核苷酸表现出独特的结构基序，因此为探测dna加工酶的催化和识别位点提供了独特的工具。以前制备位点特异性加合寡核苷酸的方法受到产率低（在50微克范围内）以及无法将加合物放置在含有多个嘌呤核苷酸残基的生物相关序列中的限制。我们已经开发出强大的新合成方法，可以在3-5毫克的范围内相对容易地合成任何所需序列的内合核苷和位点特异性内合寡核苷酸，适用于核磁共振和x射线晶体学研究以及生化研究。目前的合成工作主要集中在由二苯并[a， 1]芘二醇环氧化物（DBalP DEs）的dA和dG加合物衍生的磷酸基体的立体选择性合成，DBalP DEs是迄今为止发现的最具致癌性的多环芳烃DEs，并将其掺入寡核苷酸中。在制备双寡核苷酸的过程中，在5?-CCATCGTACC-3？序列，我们得到了令人惊讶的观察(1)，碳氢化合物部分从它与互补链共价结合的链上迁移，产生了两个新的内合产物：一个主要产物与碳氢化合物在G上互补立即与C互补5？与原加合物形成一个小产物，G上的碳氢化合物立即与C互补。到原来的加合物。在室温和中性ph下6天后，大约一半的碳氢化合物迁移到互补链上。在迁移产物中观察到碳氢化合物与DNA附着点的构型反转。多环芳烃加合物跳跃的能力双链DNA从一条链到另一条链是前所未有的，这表明了一种全新的诱变途径。我们预计目前正在进行的核磁共振研究将揭示这种独特反应的结构特征。

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) and benzo[c]phenanthrene (BcPh) are widespread environmental pollutants, many of which are potent carcinogens in mammals. These hydrocarbons are metabolically activated through the action of cytochrome P450 and epoxide hydrolase to give highly reactive bay-region diol epoxides (DEs) which form covalent DNA adducts, mainly at the exocyclic amino groups of purine bases. Erroneous replication of the modified residues by DNA polymerases leads to mutations, which represent a likely first step in the induction of cancer. Sixteen bay-region DE-purine adducts are metabolically possible for a given parent hydrocarbon. Duplex oligonucleotides containing these adducts exhibit distinct structural motifs and thus provide unique tools to probe the catalytic and recognition sites of DNA-processing enzymes. Previous methods for preparing site-specifically adducted oligonucleotides were limited by low yields (in the 50 microgram range) as well as the inability to place adducts in biologically relevant sequences containing multiple purine nucleotide residues. We have developed powerful new synthetic methods which make possible the relatively facile synthesis of adducted nucleosides and site-specifically adducted oligonucleotides in any desired sequence contest on 3-5 milligram scale, suitable for NMR and X-ray crystallographic studies as well as for biochemical investigations. Current synthetic efforts focus on stereoselective synthesis of phosphoramidite building blocks derived from dA and dG adducts of dibenzo[a,l]pyrene diol epoxides (DBalP DEs), the most carcinogenic PAH DEs identified to date, and their incorporation into oligonucleotides. In the course of preparing a duplex oligonucleotide containing a trans opened DBalP DE adduct at the central G of a 5?-CCATCGTACC-3? sequence, we made the surprising observation (1) that the hydrocarbon moiety migrated from the strand to which it was covalently bound to the complementary strand to give two new adducted products: a major product with the hydrocarbon on the G complementary to the C immediately 5? to the original adduct, and a minor product with hydrocarbon on G complementary to the C immediately 3? to the original adduct. Approximately half of the hydrocarbon had migrated to the complementary strand after six days at room temperature and neutral pH. Inversion of configuration at the point of attachment of the hydrocarbon to the DNA was observed in the migration products. The ability of a PAH adduct to ?jump? from one strand to the other in duplex DNA is unprecedented and suggests a completely new pathway for mutagenesis. We anticipate that NMR studies currently in progress on the adducted oligonucleotide duplex will reveal structural features responsible for this unique reaction. Enzymology of PAH DE Adduct Processing: Enzymes currently under study include human DNA polymerase beta, vaccinia viral topoisomerase I, Werner syndrome helicase and HIV-1 integrase. i) DNA polymerase beta (Pol beta) functions as a nucleotidyl transferase to fill gaps in one strand of double-stranded DNA that result from base excision of DNA damage followed by endonuclease cleavage at the abasic site. We have determined the crystal structure of human Pol beta bound to a 1-nucleotide gapped DNA containing a BcPh DE-deoxyguanosine (dG) adduct in the templating position opposite the gap (2). In this structure, the hydrocarbon portion of the adduct stacks over the base pair immediately 5? to the modified guanine and blocks the normal binding site for the incoming deoxynucleoside triphosphate. Thus, we observe that correct insertion of dC opposite the adduct is more than 6 orders of magnitude less efficient than opposite a normal dG template. In our structure, the modified dG assumes a syn conformation which can hydrogen bond to an incoming dATP or dGTP. Consistent with this structure, misinsertion of purine nucleotides opposite the adducted dG is preferred over insertion of the correct dC. ii) Topoisomerase I of vaccinia, a poxvirus closely related to the virus that causes smallpox, is an enzyme that relaxes supercoiled DNA. The enzyme makes a transient single-strand break in duplex DNA to give a phosphotyrosyl bond to a DNA 3'-phosphate group and a free 5?-hydroxyl group on the other side of the break. Chemically intact but relaxed DNA is formed and the free tyrosyl enzyme is regenerated in a second, religation step. In previous studies we systematically mapped topoisomerase-substrate contacts at the vaccinia enzyme?s recognition and cleavage sites by observing the effects of PAH-DE adducts of defined stereochemistry on the rate and extent of the cleavage step. BaP DE-dA adducts that intercalate into the DNA at or near the cleavage site permit cleavage, although they significantly retard its rate. We have now determined that such intercalating adducts inhibit the religation step by increasing the amount of enzyme-substrate covalent complex at equilibrium by a factor of ~10- to 15-fold, thereby trapping and ?poisoning? the topoisomerase (3). Understanding of the structural requirements for irreversible poisoning of this essential viral enzyme could lead to design of novel antiviral agents. iii) Helicases are required for unwinding of duplex DNA, and may have a role in repairing stalled replication forks in the presence of damaged DNA. Deficiencies in these enzymes result in genomic instability disorders such as Werner and Bloom syndromes. Werner syndrome helicase unwinds DNA by translocating along one strand in the 3?- to 5?-direction and displacing the opposite strand. We have found (4) that BaP dG adducts located on the strand on which the helicase translocates inhibit DNA unwinding in a manner that does not depend of their stereochemical orientation. In contrast, these dG adducts have no effect on DNA unwinding when located on the displaced strand. iv) We are continuing to explore the effects of PAH adducts and other DNA modifications on DNA cleavage and strand transfer reactions catalyzed by HIV-1 integrase. Integrase first cleaves a dinucleotide (GT) fragment from the 3?-end of one DNA strand (leaving a 2-nucleotide overhang on the opposite strand), and then catalyzes insertion of the cleaved end into another strand of DNA. We have observed (5) that intercalating BaP DE-dA adducts at or near the 3?-cleavage site of an oligonucleotide substrate have no effect on the cleavage reaction, whereas minor-groove bound BaP DE-dG adducts 1 or 2 bases 5? to the cleavage site inhibit 3?-cleavage to an extent that depends on their stereochemical orientation relative to the cleavage site. A BaP adduct on the cleaved GT dinucleotide was found to inhibit strand transfer, consistent with the interpretation that this fragment remains associated with the enzyme during the strand transfer step (5).