Mapping of DNA alkylation sites induced by adozelesin and bizelesin in human cells by ligation-mediated polymerase chain reaction

Biochemistry. 1994 May 17;33(19):6024-30. doi: 10.1021/bi00185a043.

Abstract

In this study, we have mapped the intracellular alkylation sites of adozelesin and bizelesin, two potent analogs of CC-1065, in individual genes at the single-nucleotide level. Human colon carcinoma cells were treated with adozelesin and bizelesin, and the position of adducts were mapped within the PGK-1 and p53 genes by means of ligation-mediated polymerase chain reaction. The monofunctional alkylating agent adozelesin was found to alkylate genomic DNA predominantly within 5'-(A/T)(A/T)A* sequences. Additional sites of alkylation were observed within 5'-(A/T)(G/C)(A/T)A* sequences; however, these were considered to represent sites of medium to low preference. Bizelesin, a bifunctional analog capable of both DNA monofunctional alkylation and DNA interstrand cross-link formation, was also found to alkylate 5'-(A/T)(A/T)A* sequences. Putative bizelesin DNA interstrand cross-link sites indicated that AT-rich sequences are preferred in the intervening sequence between the two cross-linked adenines. Both six- and seven-nucleotide regions were identified as putative sites of DNA interstrand cross-link formation with 5'-TTTTTTA*, 5'-TTTATCA* and 5'-GTACTAA* sequences being preferred. Non-adenine bases are not observed as potential intracellular sites of either DNA interstrand cross-linking formation or monofunctional alkylation. Thus, the patterns of alkylation induced by adozelesin and bizelesin in genomic DNA are similar but not identical to that observed in purified cell-free DNA.

Publication types

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

MeSH terms

  • Alkylating Agents / pharmacology*
  • Base Sequence
  • Benzofurans
  • Cyclohexanecarboxylic Acids / pharmacology*
  • Cyclohexenes
  • DNA / chemistry*
  • DNA / drug effects
  • Duocarmycins
  • Genes, p53
  • Humans
  • Indoles / pharmacology*
  • Molecular Sequence Data
  • Phosphoglycerate Kinase / genetics
  • Polymerase Chain Reaction / methods
  • Tumor Cells, Cultured
  • Urea / analogs & derivatives*
  • Urea / pharmacology

Substances

  • Alkylating Agents
  • Benzofurans
  • Cyclohexanecarboxylic Acids
  • Cyclohexenes
  • Duocarmycins
  • Indoles
  • adozelesin
  • Urea
  • DNA
  • Phosphoglycerate Kinase
  • bizelesin