Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase.
Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase.
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DOI：
10.1021/jacs.5b08027
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发表时间：
2015-09-23
影响因子：
15
通讯作者：
Suo Z
中科院分区：
文献类型：
--
作者：
Vyas R;Efthimiopoulos G;Tokarsky EJ;Malik CK;Basu AK;Suo Z
1-硝基芘（1-NP）是一种环境污染物，可引起体内DNA损伤，并被认为是致癌物质。由1-NP代谢物形成的DNA加合物使复制型DNA聚合酶停滞，但可能被易错的Y家族DNA聚合酶绕过，代价是体内复制保真度和效率。我们的运行启动试验证实，位点特异性放置的8-（脱氧鸟苷-N2-基）-1-氨基芘（dG 1，8），来自1-NP的DNA加合物之一，可以绕过硫磺硫化叶菌DNA聚合酶IV（DPO 4），虽然这个代表性的Y-家族酶被病变强烈暂停。采用预稳态动力学测定来确定低核苷酸掺入保真度，并建立Dpo 4旁路dG 1，8的最小动力学机制。为了揭示dCTP掺入相对dG 1，8的结构基础，我们解决了Dpo 4和DNA的复合物的晶体结构，含有模板dG 1，8病变的dCTP的存在或不存在。Dpo 4·DNA-dG 1，8二元结构显示损伤的氨基芘部分与引物/模板连接对堆叠，而其dG部分投射到Dpo 4的手指和小手指结构域之间的裂缝中。在Dpo 4·DNA-dG 1，8·dCTP三元结构中，dG 1，8损伤的氨基芘部分夹在新生和连接碱基对之间，而其碱基存在于大沟中。此外，dCTP与dG形成Watson-Crick碱基对，在dG 1，8位点上游的两个核苷酸处，产生用于“-2”移码突变的复合物。从机理上讲，这些晶体结构为上述最小动力学机制提供了额外的见解。
1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.