Lesion-specific DNA-binding and repair activities of human O⁶-alkylguanine DNA alkyltransferase.
Binding experiments with alkyl-transfer-active and -inactive mutants of human O(6)-alkylguanine DNA alkyltransferase (AGT) show that it forms an O(6)-methylguanine (6mG)-specific complex on duplex DNA that is distinct from non-specific assemblies previously studied. Specific complexes with duplex DNA have a 2:1 stoichiometry that is formed without accumulation of a 1:1 intermediate. This establishes a role for cooperative interactions in lesion binding. Similar specific complexes could not be detected with single-stranded DNA. The small difference between specific and non-specific binding affinities strongly limits the roles that specific binding can play in the lesion search process. Alkyl-transfer kinetics with a single-stranded substrate indicate that two or more AGT monomers participate in the rate-limiting step, showing for the first time a functional link between cooperative binding and the repair reaction. Alkyl-transfer kinetics with a duplex substrate suggest that two pathways contribute to the formation of the specific 6mG-complex; one at least first order in AGT, we interpret as direct lesion binding. The second, independent of [AGT], is likely to include AGT transfer from distal sites to the lesion in a relatively slow unimolecular step. We propose that transfer between distal and lesion sites is a critical step in the repair process.
Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA.
Manana
M
Melikishvili
Michael G
MG
Fried
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase