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Book of Abstracts: Albany 2009

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

DNA Repair Mutants Of O6-alkylguanine-DNA alkyltransferase (AGT) That Affect DNA Binding Affinity, Cooperativity and Repair

The O6-alkylguanine-DNA alkyltransferase (AGT) catalyzes the repair of promutagenic O6-alkylguanine and O4-alkylthymine residues in eukaryotic cells by transferring alkyl groups to residue C145 in its active site. Although one molecule of AGT occupies ˜8bp along the minor groove surface of double stranded DNA, cooperative binding to double stranded and single stranded DNAs can reach densities as high as 1 protein/4 bp (or nt). To account for these facts, we have proposed a model in which protein molecules overlap along the DNA contour. This model identifies protein surfaces that are likely to be juxtaposed in the cooperative complex. Chemical cross-linking followed by proteolysis and mass spectrometry was one method used to test the model. Consistent with predictions, the results define two protein surfaces that are adjacent in the cooperative complex but not in the free protein. Mutagenesis of residues in these surfaces has resulted in 6 mutant proteins to date, all of which fold to compact forms as measured by analytical ultracentrifugation. CD spectroscopy reveals that 3 are indistinguishable from wild-type AGT and 3 have altered secondary structure compositions. All mutant proteins have significantly reduced DNA binding constants when compared to wild type AGT in vitro. Cooperativity of DNA binding varied among the 6 mutants giving lower, similar or increased values when compared to wild type AGT. In vivo DNA repair studies using an E. coli model system showed that 3 mutants exhibit a compromised DNA repair process due to change of function rather than change in expression, while 1 mutant enhances repair due to protein over-expression. Work to correlate binding and repair activities of these mutants is under way. Supported by NIH grant GM 070662.

Claire Adams
Michael Fried

Dept of Molecular and
Cellular Biochemistry
Center for Structural Biology
University of Kentucky
Lexington, KY 40536

Tel 859 323 4613
Fax 859 257 2283
cadams@uky.edu