Kinetic and Structural Mechanisms of (5鈥?i>S)-8,5鈥?Cyclo-2鈥?deoxyguanosine-Induced DNA Replication Stalling

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• 作者：Wenyan Xu ; Adam M. Ouellette ; Zdzislaw Wawrzak ; Storm J. Shriver ; Spencer M. Anderson ; Linlin Zhao
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 27, 2015
• 年：2015
• 卷：54
• 期：3
• 页码：639-651
• 全文大小：579K
• ISSN：1520-4995

文摘

The (5鈥?i>S)-8,5鈥?cyclo-2鈥?deoxyguanosine (S-cdG) lesion is produced from reactions of DNA with hydroxyl radicals generated from ionizing radiation or endogenous oxidative metabolisms. An elevated level of S-cdG has been detected in Xeroderma pigmentosum, Cockayne syndrome, breast cancer patients, and aged mice. S-dG blocks DNA replication and transcription in vitro and in human cells and produces mutant replication and transcription products in vitro and in vivo. Major cellular protection against S-dG includes nucleotide excision repair and translesion DNA synthesis. We used kinetic and crystallographic approaches to elucidate the molecular mechanisms of S-cdG-induced DNA replication stalling using model B-family Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) and Y-family S. solfataricus P2 DNA polymerase IV (Dpo4). Dpo1 and Dpo4 inefficiently bypassed S-cdG with dCTP preferably incorporated and dTTP (for Dpo4) or dATP (for Dpo1) misincorporated. Pre-steady-state kinetics and crystallographic data mechanistically explained the low-efficiency bypass. For Dpo1, S-cdG attenuated K_d,dNTP,app and k_pol. For Dpo4, the S-cdG-adducted duplex caused a 6-fold decrease in Dpo4:DNA binding affinity and significantly reduced the concentration of the productive Dpo4:DNA:dCTP complex. Consistent with the inefficient bypass, crystal structures of Dpo4:DNA(S-cdG):dCTP (error-free) and Dpo4:DNA(S-cdG):dTTP (error-prone) complexes were catalytically incompetent. In the Dpo4:DNA(S-cdG):dTTP structure, S-cdG induced a loop structure and caused an unusual 5鈥?template base clustering at the active site, providing the first structural evidence of the previously suggested template loop structure that can be induced by a cyclopurine lesion. Together, our results provided mechanistic insights into S-cdG-induced DNA replication stalling.