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 gr oups from the O-6 position of guanine or the O-4 position of thymine b y transfering the group to an active site cysteine. In order to trap a n MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-ami nopurine (d(6)Cys(2)AP) was synthesized and incorporated into oligonuc leotides. d(6)Cys(2)AP has a disulfide bond within an alkyl chain link ed to the 6 position of 2,6-diaminopurine, which disulfide can be redu ced to form a free thiol. Addition of human MTase to reduced oligonucl eotide resulted in a protein-DNA complex that was insensitive to denat uration by SDS and high salt, but which readily dissociated in the pre sence of dithiothreitol. Formation of this complex was prevented by me thylation of the active site cysteine. Evidence that the active site c ysteine 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.