Quantitative and qualitative analysis of DNA methylation at N3-adenine by N-methyl-N-nitrosourea.
The sequence-specific alkylation of DNA by N-methyl-N-nitrosourea (MNU) has been demonstrated for the minor groove N3-methyladenine (N3-MeAde) adduct using neutral thermal hydrolysis and polyacrylamide sequencing gels. The ratio of relative yields of N7- and N3-MeAde and N7-methylguanine (N7-MeGua) is approximately 0.03:0. 15:1.00, respectively, on the basis of the gel data, and these values are comparable to relative yields determined by bulk digestion of MNU-methylated DNA when HPLC was used to analyze the individual adducts. In contrast to the methylation at N7-guanine (N7-Gua) by MNU, alkylation at Ade shows minimal sequence selectivity. Similar to the methylation at N7-Gua, formation of N3-MeAde by MNU is inhibited by 50-200 mM concentrations of NaCl and DNA binding cations, including distamycin and spermine. However, N3-MeAde formation at Ade residues within methidiumpropyl-EDTA-Fe(II) footprinted distamycin DNA affinity binding regions is selectively inhibited at low concentrations of distamycin relative to Ade sites outside of ligand binding regions, and N7-Gua within or outside the distamycin binding regions. HPLC analysis shows that distamycin also quantitatively inhibits the production of N3-methylguanine when calf thymus DNA is treated with MNU or methyl methanesulfonate. The specific inhibitory effect of distamycin, which binds in the minor groove at Ade/Thy-rich sequences, provides additional evidence that the predominant DNA lesion detected at Ade by sequencing gel analysis involves minor groove N3-MeAde modifications.
Kelly
J D
JD
Eppley Institute for Research in Cancer and Allied Diseases and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68105, USA.
Shah
D
D
Chen
F X
FX
Wurdeman
R
R
Gold
B
B
D000225:Adenine / Q000032:analysis*; D000477:Alkylating Agents / Q000737:chemistry*; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D019175:DNA Methylation; D008770:Methylnitrosourea / Q000737:chemistry*; D008969:Molecular Sequence Data; D013950:Thymus Gland / Q000737:chemistry
D000477:Alkylating Agents; D008770:Methylnitrosourea; D004247:DNA; D000225:Adenine