The release of 3-methyladenine from nucleosomal DNA by a 3-methyladenine DNA glycosylase.
Nucleosomes from chicken erythrocytes, with DNA containing an average of 144 base pairs, were alkylated with [3H]methylnitrosourea. The level of alkylation of the nucleosomal DNA was 48% of that of free DNA. The histones had approximately one tenth the radioactivity of the DNA. There was no statistically significant difference between alkylation of nucleosome bases in the major vs. minor groove. When the first 50 residues of the alkylated nucleosomal DNA were examined on sequencing gels, the 7-methylguanine and 3-methyladenine (3-MeA) residues were distributed randomly. The 3-MeA DNA glycosylase I of E. coli was used to measure the release of 3-MeA from nucleosomal DNA. Incubation at 37 degrees C resulted in a release which reached a plateau of approximately 33% of the 3-MeA groups of the nucleosomal DNA. A partially purified 3-MeA DNA glycosylase from rat liver gave similar results. The limited enzymatic release is most likely due to steric hindrance of the enzyme by the DNA-histone interactions on the surface of the core particle. An alteration of nucleosomal conformation has been suggested as an explanation for repair of nucleosomal DNA. Two model systems have been examined. The addition of ethidium bromide to alkylated nucleosomes increased the enzymatic release of 3-MeA to approximately 75% and altered the electron microscopic appearance. The chemical alkylation of nucleosomes also increased the enzymatic release of 3-MeA as well as decreased the sedimentation coefficient. All of these experiments indicate a limited availability of 3-MeA residues to the glycosylase and suggest that some conformational change must occur in vivo for complete repair.
D000225:Adenine / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D002645:Chickens; D004247:DNA / Q000097:blood*; D045647:DNA Glycosylases; D004260:DNA Repair; D004912:Erythrocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D004996:Ethidium / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008854:Microscopy, Electron; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism* / Q000648:ultrastructure
D009707:Nucleosomes; C025946:3-methyladenine; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D004996:Ethidium; D000225:Adenine