O6-methylguanine in place of guanine causes asymmetric single-strand cleavage of DNA by some restriction enzymes.
The interactions of restriction enzymes with their cognate DNA recognition sequences present a model for protein-DNA interactions. We have investigated the effect of O6-methylguanine on restriction enzyme cleavage of DNA; O6-methylguanine is a carcinogenic lesion and a structural analogue of the biological restriction inhibitor N6-methyladenine. O6-Methylguanine was synthesized into oligonucleotides at unique positions. The oligonucleotides were purified and analyzed by high-pressure liquid chromatography to assure that, within the limits of our detection, O6-methylguanine was the only modified base present. These oligonucleotides were annealed with their complement so that cytosine, and in one case thymine, opposed O6-methylguanine. DNA cleavage by restriction enzymes that recognize a unique DNA sequence, HpaII, HhaI, HinPI, NaeI, NarI, PvuII, and XhoI, was inhibited by a single O6-methylguanine in place of guanine (adenine for PvuII) within the appropriate recognition sequences. However, only the modified strand was nicked by HpaII, NaeI, and XhoI with O6-methylguanine at certain positions, indicating asymmetric strand cleavage. For all the restriction enzymes studied but AhaII, BanI, and NarI, lack of double- or single-strand cleavage correlated with inability of the O6-methylguanine-containing recognition sequence to measurably bind enzyme. None of the restriction enzymes studied were inhibited by O6-methylguanine outside their cognate recognition sequences.
Voigt
J M
JM
Lineberger Cancer Research Center, University of North Carolina Medical School, Chapel Hill 27599-7295.
Topal
M D
MD
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D004262:DNA Restriction Enzymes