Abstract
Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ωave. We found that while nearest-neighbor contacts were significant, long-range interactions dominated cooperativity and this pattern held true whether the DNA was single-stranded or duplex. Experiments with single plasmid topoisomers showed that the average cooperativity was sensitive to DNA twist, and was strongest when the DNA was slightly underwound. This suggests that AGT proteins are optimally juxtaposed when the DNA is near its torsionally-relaxed state. Most striking was the decline of binding stoichiometry with linking number. As stoichiometry and affinity differences were not correlated, we interpret this as evidence that supercoiling occludes AGT binding sites. These features suggest that AGT's lesion-search distributes preferentially to sites containing torsionally-relaxed DNA, in vivo.
Document Type
Article
Publication Date
7-7-2017
Digital Object Identifier (DOI)
https://doi.org/10.1093/nar/gkx223
Funding Information
National Institute of General Medical Sciences [GM-070662 to M.G.F.]; Department of Molecular and Cellular Biochemistry, University of Kentucky. Funding for open access charge: Department of Molecular and Cellular Biochemistry, University of Kentucky.
Related Content
Supplementary Data are available at NAR Online.
Repository Citation
Melikishvili, Manana and Fried, Michael G., "Quaternary Interactions and Supercoiling Modulate the Cooperative DNA Binding of AGT" (2017). Center for Structural Biology Faculty Publications. 8.
https://uknowledge.uky.edu/csb_facpub/8
Supplementary Data
Included in
Amino Acids, Peptides, and Proteins Commons, Genomics Commons, Molecular Biology Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Structural Biology Commons
Notes/Citation Information
Published in Nucleic Acids Research, v. 45, no. 12, p. 7226-7236.
© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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