Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic M

致癌的多环芳烃如苯并[a]芘（BaP）的海湾区二醇环氧化物（DE）代谢物被认为通过形成共价DNA加合物来启动细胞转化。两种非对映体DE，每一种都以一对对映体的形式存在，在哺乳动物中代谢形成：DE-1，其中苄基羟基和环氧氧是顺式的，DE-2，其中这些取代基是反式的。这些DE在DNA中形成的主要加合物分别由脱氧鸟苷（dG）和脱氧腺苷（dA）的环外N-2和N-6氨基顺式和反式打开环氧环而产生。我们已经开发出强大的新的合成方法，使之有可能准备网站和立体特异性DE-DNA加合物作为工具，以探测结构生物学和所需的基本细胞功能，涉及DNA加工的酶的机制酶学。这些酶包括多种DNA聚合酶，例如新发现的病变旁路聚合酶的Y超家族（见下文）、pol zeta、间隙填充聚合酶pol beta和HIV-1逆转录酶，以及拓扑异构酶、解旋酶和HIV-1整合酶。过去一年的研究主要集中在属于Y家族的聚合酶对DE-DNA损伤的处理上。与高度精确的复制型聚合酶相比，这些酶不太严重地被庞大的DNA损伤阻断，并且可以复制通过这些加合物，尽管通常以容易出错的方式。动力学研究BaP DE病变旁路的SOS诱导的Y-家族聚合酶，pols IV和V的大肠杆菌，表明，polV是唯一的SOS诱导的聚合酶能够碱基插入和延伸超出BaP DE-dA病变。它表现出有限的保真度，最常见的误差（相对于正确的T掺入约10-20%）是加合物对面的A错误掺入。对于BaP DE-dG加合物，pol V更容易出错，插入错误A的频率比正确C高350倍。相比之下，pol IV在复制过去的BaP DE-dG病变中是最有效和准确的。Pol IV也参与体内BaP DE-dG加合物的旁路：含有dG加合物的M13噬菌体在E. pol IV缺失的大肠杆菌中的表达量比野生型大肠杆菌高。杆菌我们还通过人DNA聚合酶eta研究了碱基插入和延伸对BaP DE-dG加合物的影响.这种酶是高度不准确的碱基插入相反的四个立体异构体BaP DE-dG加合物（来自顺式和反式开放的两个BaP DE-2对映异构体），并表现出强烈的偏好嘌呤错误掺入。一个可能的结构基础，这一观察涉及优先嘌呤错误插入相反的dG加合物与不寻常的顺式糖苷的方向，符合已知的顺式构象（从二维NMR研究），这样的加合物在模板引物连接。BaP DE-dG加合物构成了一个显着的块，以聚合，使进一步延伸超过加合物的网站是穷人。相比之下，酵母pol zeta（一种B家族DNA聚合酶）很容易进行延伸超过两个非对映体的反式开放BaP DE-2-dG加合物（来自DE对映体），也是高度准确的，插入正确的C对这些加合物。这表明真核聚合酶ζ在继续DNA的复制中可能起作用，DNA的延伸被Y家族聚合酶在加合物对面的碱掺入后立即阻断。

Bay-region diol epoxide (DE) metabolites of carcinogenic polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are believed to initiate cell transformation by formation of covalent DNA adducts. Two diastereomeric DEs, each of which exists as a pair of enantiomers, are formed metabolically in mammals: DE-1, in which the benzylic hydroxyl group and epoxide oxygen are cis, and DE-2, in which these substituents are trans. BaP DE adducts described in the present report are all derived from the latter diastereomer. The major adducts formed in DNA by these DEs result from cis and trans opening of the epoxide ring by the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. We have developed powerful new synthetic methods that make it possible to prepare site- and stereospecific DE-DNA adducts for use as tools to probe the structural biology and mechanistic enzymology of enzymes required for essential cellular functions involving DNA processing. These enzymes include a variety of DNA polymerases such as the newly discovered Y superfamily (see below) of lesion-bypass polymerases, pol zeta, the gap-filling polymerase pol beta, and HIV-1 reverse transcriptase, as well as topoisomerases, helicases and HIV-1 integrase. Studies during the past year have focused mainly on the processing of DE-DNA lesions by polymerases belonging to the Y family. These enzymes, in contrast to the highly accurate replicative polymerases, are less severely blocked by bulky DNA lesions and can replicate past these adducts, albeit often in an error-prone manner. Kinetic studies of BaP DE lesion bypass by the SOS-induced Y-family polymerases, pols IV and V of Escherichia coli, indicated that pol V was the only SOS-induced polymerase capable of base insertion and extension beyond BaP DE-dA lesions. It exhibited limited fidelity, with the most common error (~10-20% relative to correct T incorporation) being A misincorporation opposite the adduct. With BaP DE-dG adducts, pol V was much more error-prone and inserted an incorrect A up to 350 times more frequently than the correct C. In contrast, pol IV was the most efficient and accurate in replicating past BaP DE-dG lesions. Pol IV was also implicated in bypass of a BaP DE-dG adduct in vivo: M13 phage containing a dG adduct was replicated ~4-fold less efficiently in a mutant strain of E. coli lacking pol IV than in wild type E. coli. We have also examined base insertion and extension opposite BaP DE- dG adducts by human DNA polymerase eta. This enzyme was highly inaccurate in base insertion opposite four stereoisomeric BaP DE-dG adducts (derived from cis and trans opening of the two BaP DE-2 enantiomers), and showed a strong preference for purine misincorporation. A possible structural basis for this observation involves preferential purine misinsertion opposite dG adducts with the unusual syn glycosidic orientation, consistent with the known syn conformation (from 2-dimensional NMR studies) of such an adduct at a template-primer junction. BaP DE-dG adducts constituted a significant block to pol eta such that further extension beyond the adduct site was poor. In contrast, yeast pol zeta (a B-family DNA polymerase) readily carried out extension beyond each of the two diastereomeric trans opened BaP DE-2-dG adducts (derived from the DE enantiomers), and was also highly accurate, inserting exclusively the correct C opposite these adducts. This suggests a possible role for eucaryotic pol zeta in continuing the replication of DNA whose extension by the Y-family polymerases is blocked immediately after incorporation of a base opposite the adduct.