DESCRIPTION The goal of this project is to determine how DNA repair and replication pathways respond to the presence of the DNA adduct 8-oxoguanine (8- oxoG) in a systematically varied sequence context. The mutation frequency of damaged guanine in hotspots is likely to be governed by their flanking context.. To address this possibility, a series of sixteen oligonucleotides containing 8-oxoG flanked by all possible 5'-and 3'- based variations will be synthesized. The oligonucleotides will be placed into viral vectors for in vivo determination of mutational frequency after transfection into E. coli. Cells lacking expression of the two known 8- oxoG base excision repair enzymes, MutM and MutY, will address the impact of sequence effects on polymerase misincorporation; while cells expressing either one or both glycolyases will determine which repair system is most affected by sequence context, as determined by their relative mutation frequencies. The oligonucleotides will also be hybridized to their complements to form short duplexes for in vitro studies. The duplexes will be analyzed by thermal UV spectroscopy to determine the free energy, enthalpy and temperature of melting compared to non-lesion containing controls. The 8-oxoG containing duplexes will also be radiolabeled and digested with MutM or MutY at various concentrations to determine the rate of repair and the dissociation constant of the enzyme-substrate complex. These physiochemical data will be correlated to the mutation frequency found for each sequence context in an effort to model selective mutation or repair of oxidative DNA lesions (hot- and coldspots-respectively).
|Effective start/end date||9/1/00 → …|
- National Institutes of Health: $32,416.00
- National Institutes of Health: $40,196.00