Abstract

O6-alkylguanine DNA alkyltransferases (AGTs) are proteins found in all kingdoms of life except plants. The protein’s primary role is the removal of methyl adducts from the O6 position of 2'-deoxyguanosine (dG). However, the human protein (hAGT) has been shown to remove bulkier lesions such as benzyl groups as well inter- and intrastrand alkylene cross-links (ICLs and IaCLs, respectively) from the O6 position of dG. Repair in canonical duplex DNA has been well studied however DNA can show vast structural polymorphism. Due to its excellent programmability, ease of chemical synthesis and low cost it has been used as a nanomaterial. While initial structures were confined to two dimensions, many polyhedra have now been constructed such as tetrahedra, cubes and icosahedra based on DNA’s simple base-pairing rules. Applications of these structures have primarily been focused as a drug delivery vehicle and for biosensing. While these structures consist of the DNA double helix, DNA can fold into a variety of non-canonical structurers such as i-motifs, G-quadruplexes and triplexes. These structures have found use in nanotechnology but have also been shown to play a biological role. Modified nucleic acids that contain alternate nucleobases, backbones and sugars, which can be prepared by chemical synthesis, have been evaluated for various applications including as therapeutic agents. One example of a sugar modified nucleic acids, the threose nucleic acids (TNA) which contain a four instead of five carbon sugar have generated interest as a possible precursor to RNA in the study of origins of life.
This thesis explores four separate facets of repair in unusual nucleic acid based structures. It was investigated whether a chimeric AGT (human AGT with the active site of that of E. coli OGT) could act on IaCLs lacking a phosphate backbone as a means to covalently capture E. coli OGT’s active site. Site specific DNA-protein cross-links were prepared with dG-O6-alkylene-O6-dG and dT-O6-alkylene-O6-dG IaCLs in good yield as determined by denaturing polyacrylamide gel electrophoresis (PAGE) and electrospray ionization-mass spectrometry. A DNA tetrahedra was constructed containing dG-O6-alkylene-O6-dG three-way junctions at the vertices. These structures assembled in quantitative yield with either butylene or heptylene linkers as was assessed by PAGE under non-denaturing conditions. Furthermore, these DNA tetrahedra were well processed by human AGT at lower protein concentrations and essentially completely consumed at higher hAGT equivalents. Next, the effect of alkyl lesions at the O6 position of dG on the biophysical properties of the G-quadruplex formed by the c-myc gene promoter region was explored. Repair of these lesions by human and E. coli AGTs was also evaluated. All alkyl lesions were shown to be thermally destabilizing to the G-quadruplex independent on chain length but damage on the base-paired guanine was more destabilizing than the guanine in the loop. The E. coli AGTs were able to act on methyl lesions however struggled to process the bulkier hydroxyheptylene and IaCLs lacking a phosphate backbone. hAGT was able to process all lesions however depending on the location of the lesions variable repair efficiency was observed: Alkyl damage on the internal guanine was more poorly repaired than the external. Lastly, the synthesis of a phosphoramidite derivative of a thymine threose nucleic acid containing an O4 methyl adduct is reported. UV thermal denaturation studies of oligodeoxynucleotides containing this modification demonstrated a similar destabilizing effect to those observed with O4-methyl dT when base-paired with adenine. A similar destabilization in duplex stability was observed when mismatched with a guanine residue. Unfortunately, repair was inconclusive and further assay development is required.
These results show promise for the novel DNA tetrahedra as a means for drug delivery, cellular entry and encapsulation with further investigation ongoing. The repair of alkylation damage in the promoter region G-quadruplex of an oncogene would provide important insights with ramifications in gene regulation and structural elucidation. Conformational changes of the G-quadruplexes after being acted upon by AGTs has additional potential in nanotechnology. Future assay development on repair by AGTs on O4-methyl thymine in TNA to determine if DNA repair proteins act on TNA will provide interesting knowledge towards the chemical etiology of nucleic acid structures.