Lesion-specific DNA-binding and repair activities of human O 6-alkylguanine DNA alkyltransferase

Manana Melikishvili, Michael G. Fried

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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.

Original languageEnglish
Pages (from-to)9060-9072
Number of pages13
JournalNucleic Acids Research
Volume40
Issue number18
DOIs
StatePublished - Oct 2012

Bibliographical note

Funding Information:
National Institutes of Health (NIH) [GM-070662 to M.G.F.]; National Center for Research Resources [2P20 RR020171]. Funding for open access charge: NIH [GM-070662].

ASJC Scopus subject areas

  • Genetics

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