Interactions of human O(6)-alkylguanine-DNA alkyltransferase (AGT) with short double-stranded DNAs.
O(6)-alkylguanine-DNA alkyltransferase (AGT) is a ubiquitous enzyme with an amino acid sequence that is conserved in Eubacteria, Archaea, and Eukarya. It repairs O(6)-alkylguanine and O(4)-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 < or = omega < or = 145.0 +/- 37.0) results in an all-or-nothing association pattern on short templates. The apparent binding site size S(app) (mean = 4.39 +/- 0.02 bp) oscillates with increasing template length. Oscillations in cooperativity factor omega have the same frequency but are of opposite phase to S(app), 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 ( approximately 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.
Melikishvili
Manana
M
Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536, USA.
Rasimas
Joseph J
JJ
Pegg
Anthony E
AE
Fried
Michael G
MG
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity
D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase