Synthesis and investigation of modified oligonucleotides as probes of DNA repair

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Interstrand cross-links (ICL) can be formed in DNA as a result of endogenous processes such as lipid peroxidation and represent some of the most toxic lesions encountered by cells because they obstruct unwinding of the two strands of DNA which is critical to the processes of replication, transcription and recombination. Interference with these events in the cell by ICLs can be exploited in cancer chemotherapy regimens that employ bifunctional alkylating agents. However, the potency of these agents is diminished by the ability of cancer cells to repair the same lesions these agents induce and this causes the onset of resistance. A number of DNA repair pathways exist in cells including direct repair, base-excision repair, nucleotide-excision repair (NER), homologous recombination (HR), nonhomologous end joining and DNA-mismatch repair. Repair pathways such as NER, HR and translesion DNA synthesis (TLS) have all been implicated in ICL repair in mammalian cells, however, there is an increasing realization that ICL processing may depend on the nature of the lesion (atoms attached, linker length and orientation) making it challenging to generalize how specific ICLs are repaired. A detailed understanding of the processes by which DNA damage are recognized and repaired may lead to the design of more effective alkylating therapeutics that induce lesions that can evade repair. Our group has developed methods using a combination of solution and solid-phase synthesis to prepare sufficient quantities of ICL containing oligonucleotides for biophysical, structural and repair studies. These studies will assess whether such lesions induce a structural change in DNA that may play a role in recognition by repair enzymes. In addition, these ICL DNA will be used in experiments with DNA repair proteins to observe whether these ICL are either susceptible or evade repair processes. Correlations made between structure and repair susceptibility will aid our understanding of the processing of these therapeutically relevant lesions and will contribute to the field of DNA repair.