Synthesis of an N-methyl-N-nitrosourea linked to a methidium chloride analogue and its reactions with 32P-end-labeled DNA.
The synthesis and characterization of an N-methyl-N-nitrosourea (MNU) analogue that is covalently linked to a methidium nucleus is described. At 37 degrees C in pH 8.0 buffer 9 hydrolyzes via pseudo-first-order kinetics, with a calculated t1/2 = 77 min. By use of polyacrylamide sequencing gels the formation of piperidine-labile N7-methylguanine adducts from the reaction of 9 and MNU with 5'-32P-end-labeled DNA restriction fragments is reported. DNA methylation by 9 in 10 mM Tris buffer is enhanced with increasing ionic strength (50-200 mM NaCl), which contrasts to the inhibition of MNU-induced cleavage with increasing salt. In addition, 9 methylates all G sites equally, while MNU shows a clear preference for d(G)n (n greater than or equal to 3) runs and an asymmetrical methylation pattern within these G-rich regions. The results are discussed in terms of the delivery of the MNU moiety to the DNA target by a non-sequence-specific intercalation process and the subsequent hydrolytic generation of a nondiffusible alkylating intermediate.
Konakahara
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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68105.
Wurdeman
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RL
Gold
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B
D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism*; D005796:Genes; D005814:Genes, Viral; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008770:Methylnitrosourea / Q000138:chemical synthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D010321:Parvoviridae / Q000235:genetics; D010761:Phosphorus Radioisotopes; D011401:Promoter Regions, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics
D004279:DNA, Viral; D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; D014759:Viral Envelope Proteins; D008770:Methylnitrosourea