DNA polymerase action on an oligonucleotide containing a site-specifically located N-(deoxyguanosin-8-yl)-1-aminopyrene.
DNA polymerase action on an oligonucleotide containing a site-specifically located N-(deoxyguanosin-8-yl)-1-aminopyrene.
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DNA 聚合酶作用于含有位点特异性 N-(脱氧鸟苷-8-基)-1-氨基芘的寡核苷酸。
DOI：
10.1093/carcin/16.4.811
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发表时间：
1995
期刊：
影响因子：
4.7
通讯作者：
Basu,AK
中科院分区：
文献类型：
--
作者：
Vyas,RR;Basu,AK
合成了含有单个 N-(脱氧鸟苷-8-基)-1-氨基芘 (dGAP) 的 25 聚体寡核苷酸，dGAP 是由还原激活的 1-硝基芘形成的主要 DNA 加合物。加合物位于 3' 端的第 21 个核苷酸处。人类 DNA 聚合酶 α 和 β、HIV 逆转录酶、测序酶（2.0 版）和 DNA 聚合酶 I 的 Klenow 片段在此模板上进行的 DNA 合成在加合物位点的核苷酸 3' 处被强烈阻断。仅当使用 3'→5' 核酸外切酶缺陷的 Klenow 片段时，才会观察到与加合物相反的核苷酸的掺入。然而，超出加合物位点的延伸并未发生显着程度。仅检测到相对较小比例的全长产物（<5%）。在 Mn2+ 存在下，该聚合酶的旁路效率增加。当仅在一种三磷酸核苷酸存在的情况下延长20聚体引物时，脱氧胞苷酸优先掺入加合物对面。当一组 21mer 引物（包含与 dGAP 相对的四个核苷酸中的每一个）在所有四个脱氧核苷酸三磷酸存在下延伸为全长产物时，与加合物相对的脱氧胞苷也是优选的。为了证实这些结果，用所有四种脱氧核苷酸三磷酸进行了 15mer 引物的延伸，并分离了产物。每个延伸产物的 Maxam-Gilbert 测序表明引物延伸以无错误的方式发生。我们得出结论，dGAP 是 DNA 复制的强大阻碍。然而，当跨损伤合成发生时，它在很大程度上是准确的。
A 25mer oligonudeotide containing a singleN-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP), the major DNA adduct formed by reductively activated 1-nitropyrene, was synthesized. The adduct was located at nucleotide 21 from the 3' end. DNA synthesis on this template by human DNA polymerases α and β, HIV reverse transcriptase, Sequenase (version 2.0) and Klenow fragment of DNA polymerase I was strongly blocked at the nucleotide 3' to the adduct site. Only when a 3'→5' exonuclease-deficient Klenow fragment was used was incorporation of a nucleotide opposite the adduct observed. Nevertheless, extension beyond the adduct site did not occur to a significant extent. Only a relatively small proportion of full-length product (<5%) was detected. In the presence of Mn2+, the efficiency of bypass with this polymerase increased. When a 20mer primer was elongated in the presence of only one nucleotide triphosphate, deoxycytidylic acid was preferentially incorporated opposite the adduct. Deoxycytidine opposite the adduct was also preferred when a set of 21mer primers (containing each of the four nucleotides opposite dGAP) were elongated to a full-length product in the presence of all four deoxynucleotide triphosphates. In order to confirm these results, extension of a 15mer primer was carried out with all four deoxynucleotide triphosphates and the products were isolated. Maxam—Gilbert sequencing of each elongation product showed that primer extension occurred in an error-free manner. We conclude that dGAPis a strong block of DNA replication. However, when translesion synthesis occurs, it is largely accurate.