Modulation and Recognition of Nucleic Acid Structures

The fidelity of an organism’s genome is central to biology. DNA, however, is constantly being damaged and modified by a variety of sources. As a result of these changes, repair enzymes, polymerases, and other interrogating biomolecules must be able to recognize, repair, and adapt to a multitude of different structures and dynamics presented. Manipulation of natural systems via the development and introduction of novel bases and DNA structures only adds to this complexity. In addition, specific RNA sequences are becoming more prevalent therapeutic and diagnostic targets. These include retroviruses and other viruses that maintain their genome with RNA. Unlike DNA, RNA poses a unique challenge as targets due to their highly diverse secondary and tertiary structures. In this manuscript, three different nucleic acid systems were chosen to investigate how intramolecular and intermolecular interactions impact their own structure as well as giving further insight into how nucleic acids are recognized and distorted by interrogating damage specific enzymes as well as structure specific proteins

Synthesis of Modified Thymidine and Intrastrand Cross-Linked DNA Probes to Investigate Repair by O6-Alkylguanine DNA Alkyltransferases and Bypass by Human DNA Polymerase η

O6-Alkylguanine-DNA alkyltransferases (AGTs) are responsible for genomic maintenance by repairing O6-alkyl-2’-deoxyguanosine (O6-alkyl-dG) and O4-alkyl-thymidine (O4-alkyl-dT) adducts. AGT-mediated repair was investigated against DNA intrastrand cross-links (IaCL). A variety of cross-linked dimers linking the O6-atom of dG or O4-atom of dT were prepared synthetically to produce precursors for IaCL DNA that either lack or containing an intradimer phosphodiester group in the oligonucleotide backbone. Studies with AGTs demonstrated that: 1) O6-dG-alkylene-O6-dG flexible IaCL DNA (lacking the phosphodiester linkage) were efficiently repaired by hAGT. Repair of the model IaCL DNA occurred more efficiently in comparison to similar ICL DNA; 2) O6-dG-alkylene-O6-dG IaCL DNA containing the intradimer phosphodiester were moderately repaired by hAGT. Efficiency of the hAGT-mediated repair was contingent on the presence of the intradimer phosphate, which suggest conformational flexibility may be a requirement for repair by AGTs; 3) Flexible O4-dT-alkylene-O4-dT IaCL DNA evaded repair from all AGTs tested, whereas the flexible IaCL 5'-O4-dT-alkylene-O6-dG were efficiently repaired by hAGT. Interestingly, the 5'-O6-dG-alkylene-O4-dT was not proficiently repaired by hAGT supporting the importance of the 3'-phosphate group of the target dG nucleotide. 4) Flexible IaCL can be employed to generate DNA-protein cross-links (DPCs), with good conversions, as observed with repair of O6-dG-alkylene-O6-dG and 5'-O4-dT-alkylene-O6-dG by hAGT. The use of such cross-linking experiments may be useful for elucidating substrate discrimination across AGTs by X-ray crystallography. Translesion synthesis (TLS) may be activated by the cell as a coping mechanism when DNA damage evades repair or remains otherwise irreparable by repair mechanisms. Human DNA polymerase η (hPol η) is a key TLS Pol involved in the bypass of certain UV-induced DNA damage, and lesions formed by platinum-containing chemotherapeutics. Bypass experiments were conducted to determine if conformational freedom of the lesion impacted hPol η processivity. Towards this end, bicyclic pyrimidines linking the C5-atom to the O4-atom, by an ethylene or a propylene bridge, were synthesized as conformationally locked mimics of the biologically relevant DNA damage O4-methyl thymidine (O4-MedT) and O4-ethyl thymidine (O4-EtdT), respectively. Bypass studies revealed that: 1) The conformationally locked pyrimidyl analogues described above were bypassed by hPol η with different profiles, relative to O4-MedT and O4-EtdT. All thymidinyl modifications evoked an error-prone behavior from hPol η, with insertion of dGMP being incorporated most-frequently in the growing strand. 2) IaCL bypass profiles of O6-dG-alkylene-O6-dG containing the intradimer phosphodiester group behaved significantly different relative to those IaCL lacking it. hPol η inserted the correct nucleotide (dCMP) across the 3'-dG residue for all IaCL studied, whereas an error-prone behavior was observed across the 5'-dG residue. While the lack of the intradimer phosphodiester caused frameshift adduct formation across the 5'-dG, hPol η inserted the incorrect dTMP across the 5'-dG of the canonical IaCL DNA. More studies are required to elucidate whether this dependence is shared for other types of lesions

Probing the Activity of O6-alkylguanine-DNA Alkyltransferases on Alkylene Interstrand Cross-linked DNA

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

The design, synthesis and application of a novel electrochemical DNA gene sensor

EThOS - Electronic Theses Online ServiceGBUnited Kingdo