Interactions of human O6-alkylguanine-DNA alkyltransferase (AGT) with short double-stranded DNAs

Manana Melikishvili, Joseph J. Rasimas, Anthony Pegg, Michael G. Fried

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

O6-alkylguanine-DNA alkyltransferase ( AGT) is a ubiquitous enzyme with an amino acid sequence that is conserved in Eubacteria, Archaea, and Eukarya. It repairs O6-alkylguanine and 04-alkylthymine adducts in single-stranded and duplex DNAs. In performing these functions, AGT must partition between adduct-containing sites and the large excess of adduct-free DNA distributed throughout the genome. Here, we characterize the binding of human AGT to linear double-stranded, adduct-free DNAs ranging in length from 11 bp to 2686 bp. Moderately cooperative binding (22.6 ± 3.7 ≤ ω ≤ 145.0 ± 37.0) results in an all-or-nothing association pattern on short templates. The apparent binding site size Sapp (mean = 4.39 ± 0.02 bp) oscillates with increasing template length. Oscillations in cooperativity factor ω have the same frequency but are of opposite phase to Sapp, with the result that the most stable protein-protein and protein-DNA interactions occur at the highest packing densities. The oscillation period (4.05 ± 0.02 bp/protein) is nearly identical to the occluded binding site size obtained at the highest measured binding density (4 bp/protein) and is significantly smaller than the contour length (∼8 bp) occupied in crystalline complexes. A model in which protein molecules overlap along the DNA contour is proposed to account for these features. High AGT densities resulting from cooperative binding may allow efficient search for lesions in the context of chromatin remodeling and DNA replication.

Original languageEnglish (US)
Pages (from-to)13754-13763
Number of pages10
JournalBiochemistry
Volume47
Issue number52
DOIs
StatePublished - Dec 30 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry

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