Translesion synthesis past guanine(C8)-thymine(N3) intrastrand cross-links catalyzed by selected A- and Y-family polymerases.
Translesion synthesis past guanine(C8)-thymine(N3) intrastrand cross-links catalyzed by selected A- and Y-family polymerases.
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DOI：
10.1039/c6mb00160b
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发表时间：
2016-05-24
影响因子：
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通讯作者：
Shafirovich V
中科院分区：
文献类型：
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作者：
Lee YA;Lee YC;Geacintov NE;Shafirovich V
DNA中氧化生成的鸟嘌呤自由基可以发生各种亲核反应，包括在O2存在下与DNA中邻近或附近的N3-胸腺嘧啶形成C8-鸟嘌呤交联键。这些G[8-3]T损伤已经在暴露在氧化应激下的人类细胞的DNA中被鉴定出来，如果不通过细胞防御机制消除，很可能是遗传毒性的。几种代表性的聚合酶在两种不同的序列背景下，即G*T*和G*CT*，在体外评估了绕过G[8-3]T损伤的能力。以嗜热脂肪芽孢杆菌的聚合酶BF(杆菌片段)、硫杆菌P2的Y家族古生菌聚合酶DPO4、人DNAPOLκ和POLη为研究对象。A家族聚合酶BF被强烈阻断，而在Y家族聚合酶DPO4和POLκ中观察到相对较弱的跨损伤合成。POLη催化的引物延伸在G[8-3]T交联碱基或附近的不同位置也部分停滞，但在病变部位之外观察到显著的和分布的引物延伸，在G*CT*的情况下，效率始终高于G*T*病变的情况。POLη获得的结果与其他链内交联损伤的跨损伤合成进行了比较，其他人发表的结果包括电离辐射产生的G[8-5M]T异构体损伤，紫外线辐射产生的顺式环丁烷嘧啶二聚体和6-4光产物，以及化疗药物顺铂与DNA反应产生的铂-G*G*损伤。鸟氨酸(C8)-胸腺嘧啶(N3)链内交联链被Y-家族聚合酶以不同的效率绕过，但A-家族聚合酶BF被强烈阻断。
Oxidatively generated guanine radicals in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. These G[8-3]T lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defence mechanisms. The abilities of several representative polymerases to bypass the G[8-3]T lesions in two different sequence contexts, G*T* and G*CT* were assessed in vitro. The polymerase BF (bacillus fragment) from Bacillus stearothermophilus, the Y-family archaeal polymerases Dpo4 from Sulfolobus sulfataricus P2, and human DNA pol κ and pol η were selected for study. The A-family polymerase BF was strongly blocked, while relatively weak translesion synthesis was observed in the case of the Y-family polymerases Dpo4 and pol κ. Primer extension catalyzed by pol η was also partially stalled at various positions at or near the G[8-3]T cross-linked bases, but significant and distributive primer extension was observed beyond the sites of the lesions with the efficiency being consistently greater in the case of the G*CT* than in the case of the G*T* lesions. The results obtained with pol η are compared with translesion synthesis past other intrastrand cross-linked lesions with previously published results by others that include the isomeric G[8-5m]T lesions generated by ionizing radiation, the cis-syn cyclobutane pyrimidine dimer and the 6-4 photoproduct generated by UV radiation, and the Pt-G*G* lesions derived from the reactions of the chemotherapeutic agent cisplatin with DNA. Guanine(C8)-thymine(N3) intrastrand cross-links are bypassed with varying efficiencies by Y-family polymerases, but the A-family polymerase BF is strongly blocked.