Structural insight into dynamic bypass of the major cisplatin-DNA adduct by Y-family polymerase Dpo4

EMBO J. 2010 Jun 16;29(12):2059-69. doi: 10.1038/emboj.2010.101. Epub 2010 May 28.

Abstract

Y-family DNA polymerases bypass Pt-GG, the cisplatin-DNA double-base lesion, contributing to the cisplatin resistance in tumour cells. To reveal the mechanism, we determined three structures of the Y-family DNA polymerase, Dpo4, in complex with Pt-GG DNA. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3'G (first insertion) and 5'G (second insertion) of Pt-GG, and the primer extension beyond the lesion site. We observed a dynamic process, in which the lesion was converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4. The DNA translocation-coupled conformational change may account for additional inhibition on the second insertion reaction. The structures illustrate that Pt-GG disturbs the replicating base pair in the active site, which reduces the catalytic efficiency and fidelity. The in vivo relevance of Dpo4-mediated Pt-GG bypass was addressed by a dpo-4 knockout strain of Sulfolobus solfataricus, which exhibits enhanced sensitivity to cisplatin and proteomic alterations consistent with genomic stress.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Catalytic Domain
  • Cell Survival
  • Cisplatin / chemistry
  • Cisplatin / metabolism*
  • Cisplatin / pharmacology
  • Crystallography, X-Ray
  • DNA Adducts / chemistry
  • DNA Adducts / metabolism*
  • DNA, Archaeal / biosynthesis*
  • DNA, Archaeal / chemistry
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Electrophoresis, Gel, Two-Dimensional
  • Gene Knockout Techniques
  • Models, Molecular
  • Protein Structure, Tertiary
  • Proteome / analysis
  • Sulfolobus solfataricus / chemistry
  • Sulfolobus solfataricus / genetics
  • Sulfolobus solfataricus / metabolism*

Substances

  • Antineoplastic Agents
  • Archaeal Proteins
  • DNA Adducts
  • DNA, Archaeal
  • Proteome
  • cisplatin-DNA adduct
  • DNA-Directed DNA Polymerase
  • Cisplatin

Associated data

  • PDB/3M9M
  • PDB/3M9N
  • PDB/3M9O