Kinetic Investigation of Translesion Synthesis across a 3-Nitrobenzanthrone-Derived DNA Lesion Catalyzed by Human DNA Polymerase Kappa
Kinetic Investigation of Translesion Synthesis across a 3-Nitrobenzanthrone-Derived DNA Lesion Catalyzed by Human DNA Polymerase Kappa
复制标题
人类 DNA 聚合酶 Kappa 催化的 3-硝基苯并蒽酮衍生 DNA 损伤跨损伤合成的动力学研究
DOI：
10.1021/acs.chemrestox.9b00219
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发表时间：
2019
影响因子：
4.1
通讯作者：
Suo, Zucai
中科院分区：
文献类型：
--
作者：
Phi, Kenneth K.;Smith, Madison C.;Tokarsky, E. John;Suo, Zucai
3-硝基苯蒽酮（3-NBA）是柴油机废气的副产品，在工业和人口密集地区大量存在。吸入3-NBA会导致N-（2′-脱氧鸟苷-8-基）-3-氨基苯并蒽酮（dGC 8-N-ABA）的形成，这是一种体积庞大的DNA损伤，由于其潜在的致突变性和致癌性而受到关注。如果dGC 8-N-ABA在基因组复制过程中没有被绕过，则损伤可以使细胞DNA复制机器停止，导致衰老或凋亡。我们之前已经使用运行启动测定来证明人DNA聚合酶eta（hPolη）和kappa（hPolκ）能够催化跨DNA模板中的位点特异性放置的dGC 8-N-ABA的跨损伤DNA合成（TLS）。一致地，HEK 293 T细胞中hPolη和hPolκ的基因敲低分别使TLS穿过dGC 8-N-ABA的效率降低了25%和30%。在这里，我们动力学研究了为什么hPolκ在绕过dGC 8-N-ABA并从dGC 8-N-ABA延伸时暂停。我们的动力学数据显示，相对于未受损的dG，当与dGC 8-N-ABA相反时，hPolκ的正确dCTP掺入效率下降了116倍，导致在运行开始测定中观察到的损伤部位处的hPolκ暂停。hPolκ已经很低的核苷酸掺入保真度在病变旁路期间进一步降低了10倍，因此，不正确的核苷酸，特别是dATP，以与正确的dCTP相当的效率相对于dGC 8-N-ABA掺入。关于dGC 8-N-ABAbypass产物延伸步骤，hPolκ将正确的dGTP掺入到受损的DNA底物上的效率比掺入到相应的未受损的DNA底物上的效率低786倍，这导致hPolκ在运行开始测定中在该位点暂停。此外，hPolκ延伸引物末端匹配的碱基对dC：dGC 8-N-ABA的保真度比其延伸未受损的dC：dG碱基对低100-1000倍。总之，我们的动力学结果强烈表明，hPolκ在dGC 8-N-ABA的TLS期间是易错的。
3-Nitrobenzanthrone (3-NBA) is a byproduct of diesel exhaust and is highly present in industrial and populated areas. Inhalation of 3-NBA results in formation ofN-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of concern due to its mutagenic and carcinogenic potential. If dGC8-N-ABAis not bypassed during genomic replication, the lesion can stall cellular DNA replication machinery, leading to senescence or apoptosis. We have previously used running start assays to demonstrate that human DNA polymerases eta (hPolη) and kappa (hPolκ) are able to catalyze translesion DNA synthesis (TLS) across a site-specifically placed dGC8-N-ABAin a DNA template. Consistently, gene knockdown of hPolη and hPolκ in HEK293T cells reduces the efficiency of TLS across dGC8-N-ABAby ∼25 and ∼30%, respectively. Here, we kinetically investigated why hPolκ paused when bypassing and extending from dGC8-N-ABA. Our kinetic data show that correct dCTP incorporation efficiency of hPolκ dropped by 116-fold when opposite dGC8-N-ABArelative to undamaged dG, leading to hPolκ pausing at the lesion site observed in the running start assays. The already low nucleotide incorporation fidelity of hPolκ was further decreased by 10-fold during lesion bypass, and thus, incorrect nucleotides, especially dATP, were incorporated opposite dGC8-N-ABAwith comparable efficiencies as correct dCTP. With regard to the dGC8-N-ABAbypass product extension step, hPolκ incorporated correct dGTP onto the damaged DNA substrate with a 786-fold lower efficiency than onto the corresponding undamaged DNA substrate, which resulted in hPolκ pausing at the site in the running start assays. Furthermore, hPolκ extended the primer-terminal matched base pair dC:dGC8-N-ABAwith a 100–1000-fold lower fidelity than it extended the undamaged dC:dG base pair. Together, our kinetic results strongly indicate that hPolκ was error-prone during TLS of dGC8-N-ABA.