Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase
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- 作者：Rajan Vyas ; Georgia Efthimiopoulos ; E. John Tokarsky ; Chanchal K. Malik ; Ashis K. Basu ; Zucai Suo
- 刊名：Journal of the American Chemical Society
- 出版年：2015
- 出版时间：September 23, 2015
- 年：2015
- 卷：137
- 期：37
- 页码：12131-12142
- 全文大小：760K
- ISSN：1520-5126
文摘
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-N<sup>2sup>-yl)-1-aminopyrene (dG<sup>1,8sup>), 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 dG<sup>1,8sup> bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG<sup>1,8sup>, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG<sup>1,8sup> lesion in the absence or presence of dCTP. The Dpo4路DNA-dG<sup>1,8sup> 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路DNA-dG<sup>1,8sup>路dCTP ternary structure, the aminopyrene moiety of the dG<sup>1,8sup> lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson鈥揅rick base pair with dG, two nucleotides upstream from the dG<sup>1,8sup> site, creating a complex for 鈥?2鈥?frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.