Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Substrate Interactions of a Human DNA Alkyltransferase
Human cells contain DNA alkyltransferases that protect genomic integrity under normal conditions but also defend tumor cells against chemotherapeutic alkylating agents. Here we explore how structural features of the DNA substrate affect the binding and repair activities of the human O6-alkylguanine-DNA alkyltransferase (AGT).
To perform its repair functions, AGT partitions between adduct-containing sites and the large excess of adduct-free genomic DNA. Cooperative binding 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 template length. Oscillations in cooperativity factor ω have the same frequency but are of opposite phase to Sapp so the most stable complexes occur at the highest packing densities. At high binding densities the site size (~4 bp/protein) is much smaller than the contour length (~8 bp) occupied in crystalline 1:1 complexes. A model in which protein molecules overlap along the DNA contour has been proposed; this model predicts that optimal protein-protein contacts will occur when the DNA is torsionally relaxed. Supporting this prediction, competition assays show that AGT binds relaxed DNAs in preference to negatively-supercoiled forms and topoisomerase assays show that AGT binding is accompanied by a small-but-measurable net unwinding (7.14 ± 0.33 deg/protein). These results predict that AGT will partition in favor of torsionally-relaxed, relatively protein-free DNA structures like those near replication forks.
AGT must also function at telomeres, where G-rich sequences have the potential to form quadruplex structures and where methylation at the O6 position of guanines interferes with quadruplex formation. AGT interactions with small unimolecular quadruplexes are characterized by reduced binding stoichiometries, affinities and O6-methyl G repair activities when compared to those with linear DNAs. Thus, AGT may function best at telomeres when quadruplex formation is inhibited by helicases or other telomere-binding proteins.
Model of AGT-DNA complex with double-stranded DNA. The repeating unit of this model is one molecule of AGT (colors) plus 4 base-pairs of DNA (black); the coordinates were derived from PDB file 1T38. Repeating units were juxtaposed with preservation of B-DNA helical parameters (separation =3.4 Å, twist = 34.6 degrees) between base-pairs of adjacent units. The result is a 3-start helical array (left) with important contacts between proteins n and n + 3 (right).
This work was supported by NIH grant GM070662 to MGF.
Department of Molecular and Cellular Biochemistry
University of Kentucky
741 S. Limestone St., Lexington, KY 40536