Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.
DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.
Paalman
S R
SR
Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
Noll
D M
DM
Clarke
N D
ND
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004220:Disulfides / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D013438:Sulfhydryl Compounds / Q000032:analysis
D003432:Cross-Linking Reagents; D004220:Disulfides; D009943:Organophosphorus Compounds; D013438:Sulfhydryl Compounds; D006147:Guanine; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine