Kinetic and structural mechanisms of (5′S)-8,5′-cyclo-2′-deoxyguanosine-induced DNA replication stalling

Wenyan Xu, Adam M. Ouellette, Zdzislaw Wawrzak, Storm J. Shriver, Spencer M Anderson, Linlin Zhao*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

The (5′S)-8,5′-Cyclo-2′-deoxyguanosine (ScdG) 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 Kd,dNTP,app and kpol. 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( ScdG): 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′-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. (Graph Presented)

Original languageEnglish (US)
Pages (from-to)639-651
Number of pages13
JournalBiochemistry
Volume54
Issue number3
DOIs
StatePublished - Jan 27 2015

ASJC Scopus subject areas

  • Biochemistry

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