Investigation of the Substrate Range of O6-Alkylguanine-DNA Alkyltransferases Using Chemically Modified Oligonucleotides and the Properties of Parallel Stranded Adenosine Duplexes
Dr. Christopher Wilds
Department of Chemistry and Biochemistry
Friday, December 6, 2013
Abstract: O6-alkylguanine-DNA alkyltransferases (AGT) are responsible for the removal of mutagenic O6-alkyl 2’-deoxyguanosine and O4-alkyl thymidine adducts. AGT homologues show vast substrate differences with respect to the size of the adduct and which alkylated atoms they can restore. Our group has been interested in exploring AGTs ability to remove various lesions in DNA including interstrand cross-links (ICL). Oligonucleotide probes containing alkylene linkers varying in length that tethered the O6 atoms of 2’-deoxyguanosine (dG) or O4 atoms of thymidine (T) have been prepared using a combination of solution and solid phase synthesis. [2,3] Studies with the AGTs and the modified DNA probes have revealed that human AGT is capable of repairing O6-2’-deoxyguanosine-alkylene- O6-2’-deoxyguanosine ICLs in a 5’-GNC sequence motif whereas O4-thymidine-alkylene-O4-thymidine ICLs evade repair.
In the second part of my presentation, I will also discuss the structure and properties of a parallel stranded duplex formed by an adenosine undecamer (rA11). Over 50 years ago, Rich and coworkers described the structure of a polyadenylic acid (poly rA) duplex at low pH based on fiber diffraction patterns.  They proposed a double helix structure where the strands were parallel in orientation with protonation of the N1 atom of adenine stabilizing the duplex. Recently, we were able to obtain crystals of rA11, grown in the presence of a fragment of the RNA binding protein PABPC1 at neutral pH (7).  This structure reveals that ammonium cations complex the RNA phosphates and N1 atom of adenine. The contribution of the ammonium cations to the stabilization of this duplex was further evaluated by UV thermal denaturation experiments. At ammonium concentrations of 4 M, sigmoidal transitions were observed at pH 7.0 for poly rA and may offer an additional structural motif that may find applications in nucleic acid nanotechnology.
 Pegg, A. E. Mutation research 2000, 462, 83.
 McManus, F. P.; Fang, Q.; Booth, J. D.; Noronha, A. M.; Pegg, A. E.; Wilds, C. J. Org Biomol Chem 2010, 8, 4414.
 McManus, F. P.; Khaira, A.; Noronha, A. M.; Wilds, C. J. Bioconjugate Chem 2013, 24, 224.
 Rich, A.; Davies, D. R.; Crick, F. H.; Watson, J. D. Journal of molecular biology 1961, 3, 71.
 Safaee, N.; Noronha, A. M.; Rodionov, D.; Kozlov, G.; Wilds, C. J.; Sheldrick, G. M.; Gehring, K. Angew Chem Int Ed Engl 2013, 52, 10370.