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Abstract
Binding experiments with alkyl-transfer-active and -inactive mutants of human O6-alkylguanine DNA alkyltransferase (AGT) show that it forms an O6-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.
Document Type
Article
Publication Date
2012
Digital Object Identifier (DOI)
http://dx.doi.org/10.1093/nar/gks674
Repository Citation
Melikishvili, Manana and Fried, Michael G., "Lesion-Specific DNA-Binding and Repair Activities of Human O⁶-Alkylguanine DNA Alkyltransferase" (2012). Center for Structural Biology Faculty Publications. 6.
https://uknowledge.uky.edu/csb_facpub/6
Notes/Citation Information
Published in Nucleic Acids Research, v. 40, no. 18, p. 9060-9072.
© The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.