19th-banner-rev.gif

Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

Sequence- And Structure-Specific DNA Base-Flipping By AGT

Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transfer of alkyl-groups from the O6 positions of guanine residues and O4 positions of thymine residues to residue C145 in its active site. This process involves a conformation change in which a DNA base becomes extrahelical and is bound within the active site of the protein. To characterize this conformational change, we have carried out hydroxyl radical (·OH) footprinting on oligonucleotide substrates of different length, base-composition and secondary structures. In AGT complexes with single-stranded DNAs, one AGT molecule protects at least three bases from ·OH, but potential cleavage sites flanking Guanine residues were hypersensitive to attack. Duplex DNAs, including sequences containing Guanine, were far less susceptible to attack by ·OH. We propose that this reflects the 2-fold degeneracy of duplex DNA and the corresponding AGT complexes but also a reduction in base un-stacking in duplex DNA. Changes in DNA circular dichroism and in the fluorescence of 2-amino purine labeled DNA support these conclusions. EMSA results with the same DNAs show that AGT binds preferentially to double-stranded DNAs and that the binding site sizes are slightly smaller and binding cooperativity higher on binding to duplex DNAs than on binding ssDNAs. These results suggest mechanisms by which AGT may search and access alkylated DNA bases for repair. Supported by NIH grant GM070662.

Manana Melikishvili
Michael G. Fried

Department of Molecular and
Cellular Biochemistry
University of Kentucky
Lexington, KY 40536-0509

ph: 859-323-4613
Fax: 859-257-2283
Email: mmeli2@uky.edu