Chenna A, Hang B, Rydberg B, Kim E, Pongracz K, Bodell W J, Singer B
Life Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720, USA.
Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5890-4. doi: 10.1073/pnas.92.13.5890.
Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.
苯是一种普遍存在于人类环境中的致癌物。其主要代谢产物之一是对苯二酚，对苯二酚在体内被氧化生成对苯醌（p-BQ）。这两种代谢产物对人体细胞均有毒性。p-BQ与DNA反应，与脱氧胞苷、脱氧腺苷和脱氧鸟苷形成苯并环丁烷加合物。在本研究中，我们分别通过使脱氧胞苷和脱氧腺苷与p-BQ反应，合成了环外化合物3-羟基-3-N4-苯并环丁烷-2'-脱氧胞苷（p-BQ-dCyd）和9-羟基-1,N6-苯并环丁烷-2'-脱氧腺苷（p-BQ-dAdo）。将它们转化为亚磷酰胺，然后用于制备在两个不同的特定位置带有p-BQ-dCyd或p-BQ-dAdo加合物（序列中的pbqC或pbqA）的位点特异性寡核苷酸。这些寡核苷酸在加合物的5'端被部分纯化的HeLa细胞提取物有效切割——含pbqC的寡聚物比含pbqA的寡聚物切割得更快。与氯乙烯代谢产物氯乙醛产生的衍生物与酶的结合情况相反，在凝胶阻滞试验中，含有p-BQ加合物的长达60聚体的寡核苷酸与相同的酶制剂没有可测量的结合。此外，即使在120倍过量的情况下，含有1,N6-乙烯基腺嘌呤脱氧腺苷（1,N6-乙烯基-dAdo；序列中的εA）的寡核苷酸与这些含有p-BQ加合物之一的寡聚物之间也没有观察到结合竞争。当获得高度纯化的快速蛋白质液相色谱（FPLC）酶组分时，似乎有两种紧密洗脱的切割活性。其中一种酶结合并切割含εA的脱氧寡核苷酸。另一种酶切割含pbqA和pbqC的脱氧寡核苷酸。另一个意外发现是，大量p-BQ处理的鲑鱼精子DNA确实能与含εA的寡核苷酸有效竞争蛋白质结合。这增加了这样一种可能性，即这种DNA含有其他尚未表征的加合物，这些加合物能被识别乙烯基加合物的同一种酶识别。总之，我们描述了一种以前未描述过的人类DNA修复活性，可能是一种糖基化酶，它能从DNA中切除pbqC和pbqA，这是脱氧胞苷和脱氧腺苷与苯代谢产物p-BQ反应产生的环外加合物。这种糖基化酶活性与本实验室先前报道的从DNA中切除四种乙烯基碱基的活性不同。
