The purine 2-amino group as the critical recognition element for sequence-specific alkylation and cross-linking of DNA by mitomycin C

Maria Tomasz, Arunangshu Das, Kit S. Tang, Marjin G.J. Ford, Andrew Minnock, Steven M. Musser, Michael J. Waring

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Abstract

Mitomycin C (MC) is a G·C-specific antitumor antibiotic that alkylates and cross-links DNA at 2-amino groups of guanine residues. Both reactions are known to be enhanced at guanines in the CpG sequence by independent mechanisms. The mechanisms were probed by substituting 2,6-diaminopurine (D) into oligonucleotides and into a 162-bp tyrT DNA restriction fragment and determining their alkylation and crosslinking by MC. Covalent D-MC adducts were isolated and structurally characterized. The results indicated that 2,6- diaminopurine functioned as a substrate analogue of guanine and displayed enhanced reactivity toward MC in all systems. The observed TpD sequence selectivity of the modifications by MC was analogous to the CpG sequence selectivity of guanine modifications. Selective monoalkylation and cross- linking was observed also at the TpG·CpD sequence, indicating that two purine 2-amino groups are necessary and sufficient for the selectivity regardless of whether they are supplied by G or by D. These findings reinforce the previously proposed mechanism in which the selectivity of monoalkylation by MC is attributed to a specific H-bond between the drug and the 2-amino group of a guanine. The specific sequence required for D-D and D- G cross-link formation was established as TpD·TpD and TpG·CpD, respectively, determined by the same minor groove structural factors as in the CpG·CpG cross-linking process. MC cross-linking was also probed in a 162-bp tyrT DNA fragment in which Gs and As were replaced by inosine and 2,6- diaminopurine, respectively, using PCR. Cross-linked sites were quantitated and mapped on the basis of an empirical correlation between the electrophoretic mobility of cross-linked DNA and the position of the cross- links relative to the center of the sequence. In the natural DNA sequence hotspots for formation of MC-cross-links were identified. The cross-links were shown to be translocated from CpG·CpG in the natural DNA to TpD·TpD or TpG·CpD in the substituted DNAs, demonstrating the dominant role of the purine 2-amino group for cross-link site selection by MC.

Original languageEnglish (US)
Pages (from-to)11581-11593
Number of pages13
JournalJournal of the American Chemical Society
Volume120
Issue number45
DOIs
Publication statusPublished - Nov 18 1998

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All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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