Interactions of human O6-alkylguanine-DNA alkyltransferase (AGT) with short single-stranded DNAs.
The O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. Here we characterize the binding of AGT to single-stranded DNAs ranging in length from 5 to 78 nucleotides (nt). Binding is moderately cooperative (37.9 +/- 3.0 <or= omega <or= 89.8 +/- 8.9), resulting in an all-or-nothing association pattern on short templates. This cooperativity contrasts with the isolated binding seen in recent crystal structures of AGT-DNA complexes. The statistical binding site size S (mean = 5.2 +/- 0.1) oscillates with increasing template length. The oscillation period (4.10 +/- 0.02 nt/protein) is nearly identical to the binding site size obtained at the highest known binding density (S = 4 nt/protein) and is significantly smaller than the contour length (approximately 8 bp) occupied in crystalline complexes. A model in which AGT proteins overlap along the DNA contour is proposed to account for these features. Oscillations in intrinsic binding constant Ki and cooperativity factor omega have the same frequency but are of opposite phase to S with the result that the most stable protein-protein and protein-DNA interactions occur at the highest packing densities. We hypothesize that modest binding cooperativity and high binding densities are adaptations that allow AGT to efficiently search for lesions in the context of chromatin remodeling and DNA replication.
Rasimas
Joseph J
JJ
Department of Molecular Physiology, Penn State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Kar
Sambit R
SR
Pegg
Anthony E
AE
Fried
Michael G
MG
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D011994:Recombinant Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase