Quaternary interactions and supercoiling modulate the cooperative DNA binding of AGT

Manana Melikishvili, Michael G. Fried

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


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.

Original languageEnglish
Pages (from-to)7226-7236
Number of pages11
JournalNucleic Acids Research
Issue number12
StatePublished - Jul 1 2017

Bibliographical note

Funding Information:
National Institute of General Medical Sciences [GM- 070662 to M.G.F.]; Department of Molecular and Cellular Biochemistry, University ofKentucky. Funding for open access charge: Department of Molecular and Cellular Biochemistry, University of Kentucky.

Publisher Copyright:
© The Author(s) 2017.

ASJC Scopus subject areas

  • Genetics


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