The release of 3-methyladenine from nucleosomal DNA by a 3-methyladenine DNA glycosylase

Nucleosomes from chicken erythrocytes, with DNA containing anaverage of 144 base pairs, were alkylated with [³H]methylnitrosourea.The level of alkylation of the nucleo-somal DNA was 48% of thatof free DNA. The histones had approximately one tenth the radioactivityof the DNA. There was no statistically significant differencebetween alkylation of nucleosome bases in the major vs. minorgroove. When the first 50 residues of the alkylated nudeosomalDNA were examined on sequencing gels, the 7-methylguanine and3-methyladenine (3-MeA) residues were distributed randomly.The 3-MeA DNA glycosylase I of E. coli was used to measure therelease of 3-MeA from nudeosomal DNA. Incubation at 37°Cresulted in a release which reached a plateau of {small tilde}33%of the 3-MeA groups of the nudeosomal DNA. A partially purified3-MeA DNA glycosylase from rat liver gave similar results. Thelimited enzymatic release is most likely due to steric hindranceof the enzyme by the DNA-histone interactions on the surfaceof the core partide. An alteration of nudeosomal conformationhas been suggested as an explanation for repair of nudeosomalDNA. Two model systems have been examined. The addition of ethidiumbromide to alkylated nudeosomes increased the enzymatic releaseof 3-MeA to {small tilde}75% and altered the electron microscopicappearance. The chemical alkylation of nudeosomes also increasedthe enzymatic release of 3-MeA as well as decreased the sedimentationcoefficient. AD of these experiments indicate a limited availabilityof 3-MeA residues to the glycosylase and suggest that some conformationalchange must occur in vivo for complete repair.