Repair of cisplatin--DNA adducts by the mammalian excision nuclease

Biochemistry. 1996 Aug 6;35(31):10004-13. doi: 10.1021/bi960453+.


Nucleotide excision repair is one of the many cellular defense mechanisms against the toxic effects of cisplatin. An in vitro excision repair assay employing mammalian cell-free extracts was used to determine that the 1,2-d(ApG) intrastrand cross-link, a prevalent cisplatin-DNA adduct, is excised by the excinuclease from a site-specifically modified oligonucleotide 156 base pairs in length. Repair of the minor interstrand d(G)/d(G) cross-link was not detected by using this system. Proteins containing the high mobility group (HMG) domain DNA-binding motif, in particular, rat HMG1 and a murine testis-specific HMG-domain protein, specifically inhibit excision repair of the intrastrand 1,2-d(GpG) and -d(ApG) cross-links. This effect was also exhibited by a single HMG domain from HMG1. Similar inhibition of repair of a site-specific 1,2-d(GpG) intrastrand cross-link by an HMG-domain protein also occurred in a reconstituted system containing highly purified repair factors. These results indicate that HMG-domain proteins can block excision repair of the major cisplatin-DNA adducts and suggest that such an activity could contribute to the unique sensitivity of certain tumors to the drug. The reconstituted excinuclease was more efficient at excising the 1,3-d(GpTpG) intrastrand adduct than either the 1,2-d(GpG) or d(ApG) intrastrand adducts, in agreement with previous experiments using whole cell extracts [Huang, J. -C., Zamble, D. B., Reardon, J. T., Lippard, S. J., Sancar, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394-10398]. This result suggests that structural differences among the platinated DNA substrates, and not the presence of unidentified cellular factors, determine the relative excision repair rates of cisplatin-DNA intrastrand cross-links in the whole cell extracts.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Cholesterol / metabolism
  • Cisplatin*
  • DNA Adducts*
  • DNA Repair*
  • Endodeoxyribonucleases / metabolism*
  • HeLa Cells
  • High Mobility Group Proteins / chemistry
  • High Mobility Group Proteins / metabolism
  • High Mobility Group Proteins / pharmacology*
  • Humans
  • Kinetics
  • Male
  • Mammals
  • Mice
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Substrate Specificity
  • Testis


  • DNA Adducts
  • High Mobility Group Proteins
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • cisplatin-DNA adduct
  • Cholesterol
  • Endodeoxyribonucleases
  • Cisplatin