Abstract

O6-Alkylguanine-DNA alkyltransferases (AGT) are responsible for the removal of numerous mutagenic O6-alkyl 2’-deoxyguanosine (O6-alkyl dG) and O4-alkyl thymidine (O4-alkyl dT) adducts. The function of AGT in the recognition and removal of both mono-adducts and DNA interstrand cross-links (ICL) were undertaken. To achieve this, oligonucleotide probes containing alkylene linkers varying in length that tethered the O4 atoms of thymidine (dT) or 2’-deoxyuridine (dU) and O6 atoms of 2’-deoxyguanosine (dG) were prepared. These ICL were generated using a combination of solution and solid phase synthesis. Various O4-alkyl dT and O4-alkyl dU mono-adducts were also prepared by similar methods and analyzed to further elucidate the limitation of AGT with respect to damage detection and repair. Three AGT homologues (human AGT/hAGT, and E. Coli OGT and Ada-C) and an engineered chimera were used in repair and binding studies with the O4 and O6 modified oligonucleotides to note variations amongst species. Studies with the AGTs and modified DNA probes revealed: 1) hAGT is capable of repairing O6-2’-deoxyguanosine-butylene-O6-2’-deoxyguanosine (O6dG-butylene-O6dG) and O6-2’-deoxyguanosine-heptylene-O6-2’-deoxyguanosine (O6dG-heptylene-O6dG) ICLs in a 5’-GNC sequence motif, designed to mimic the configuration of the ICL generated by hepsulfam (1,7-disulfamoyloxyheptane); 2) O4-thymidine-alkylene-O4-thymidine (O4dT-alkylene-O4dT) ICLs evade repair from all AGTs studied, whereas the O4-butyl-4-ol and O4-heptyl-7-ol dT mono-adducts undergoes repair by OGT; 3) Creating an hAGT:O4-alkyl dT covalent complex for crystallography by employing O6-2’-deoxyguanosine-alkylene-O4-thymidine (O6dG-alkylene-O4dT) ICL DNA shows promise since covalent complex formation is observed with O6dG-heptylene-O4dT cross-links; 4) Introducing the active site of OGT into the hAGT scaffold confers the chimera with enhanced repair capabilities of O4-alkyl dT adducts with respect to hAGT while maintaining ICL repair activity; 5) AGT mediated repair of O4-alkyl dT damage is hindered by the presence of the C5 methyl (up to 30-fold). This effect is most notable with hAGT and the chimera; 6) O4-2’-deoxyuridine-alkylene-O4-2’-deoxyuridine (O4dU-alkylene-O4dU) ICL DNAs are not repaired by AGT. This elusive property is attributed to the E conformation adopted by the linker about the C4-O4 bond of the modified dU, as demonstrated from NMR studies. There are no working models that provide a feasible hAGT based repair mechanism for ICL damage. ICL DNA:hAGT crystal structures are needed to shed light on this poorly understood activity.