Interstrand cross-links (ICLs) are covalent linkages formed by reaction of nucleobases with a variety of bifunctional agents of exogenous (e.g. nitrogen mustard reagents) and endogenous (e.g. malondialdehyde) origin. Recently, electrophilic DNA lesions such as an AP site (apurinic/apyrimidinic site), C4;;-oxidized abasic site (C4-AP), and dioxobutane (DOB) have also been implicated in ICL formation. A covalent linkage between the DNA strands prevents their separation, acting as a block for necessary cellular processes such as transcription and replication. Given the deleterious consequences of ICL formation on cellular pathways, eukaryotes and prokaryotes use nucleotide excision repair (NER) to remove interstrand cross-links. On one hand, cross-link-causing external agents, a number of which are active anti-cancer drugs, try to exploit the toxicity of ICLs to kill the cell. On the other hand, diseases that hamper activities of proteins involved in NER (e.g. Fanconi anemia) increase vulnerability to ICL-causing agents. Therefore, understanding the mechanism of cross-link formation and tracing their biochemical fate remains extremely important.C4-AP and DOB lesions are formed by anti-cancer chemotherapeutics and gama-irradiation. The lesions reversibly react with the N6 amine of an adenosine (A) to form an unstable ICL. While the ICL between C4-AP and A has 3 h half-life, the one between DOB and A is modestly more stable (t1/2 = 10.1 h). Although unstable, both ICLs last long enough to potentially disrupt replication and transcription in cells. Intriguingly, both C4-AP and DOB ICLs are formed in a site selective manner, with an A opposite their 3;;-adjacent thymidine (T). The distances computed between N6 amine of A at the preferred site and the C1;;-aldehyde of the electrophilic lesions is not radically different from that involving an opposing A, making the site selectivity ambiguous. The C4-AP ICL eventually reverts to the lesion, undergoes a strand scission and forms a thermodynamic cross-link. The short lifetime of DOB and C4-AP ICLs make the investigation of their biochemical fate and site selective formation difficult.Herein, we report the development of synthetic methodologies to prepare stable analogues of DOB and C4-AP ICLs. The procedures involve orthogonal removal of protecting groups from oligonucleotides on solid support without affecting any native DNA nucleobase or cyanoethyl protection and subsequent solid phase synthesis. In addition to being able to prepare any cross-link irrespective of sequence, the strategies can be applied to the synthesis of branched DNA and RNA.The stable analogues of C4-AP and DOB ICLs were treated with UvrABC, the bacterial nucleotide excision repair proteins to understand their repair. The incision on C4-AP lesion was slow, with maximum 27 – 30% cleavage reported after 8 h, implying potentially deleterious consequence for processes like transcription and replication. No double strand break caused by repetitive incision on opposing strand was detected. In contrast, the DOB ICL analogue exclusively produced double strand breaks upon UvrABC treatment.In addition to distance, relative stability in resulting ICL secondary structure was hypothesized as a driving force behind site selective cross-link formation. Stable oligonucleotides analogues of DOB and C4-AP ICLs with ;;observed” (ICL with A opposite 3;;-adjacent T) and ;;unobserved” (ICL with opposing A) connectivities were synthesized. Upon treatment with chemical agents that detect distorted (solvent exposed) nucleotides, the oligonucleotides with ;;unobserved” ICLs were found to have greater distortion. This was further verified using thermodynamic UV melting, where the ;;observed” ICLs had higher Tms.In conclusion, our work describes novel strategies for synthesizing stable analogues of DOB and C4-AP ICLs irrespective of their sequence. The work is compatible with other ICL preparations, but is also extendable to branched DNA and RNA synthesis. The stable analogues were employed to address the biochemical fate of the respective ICLs in the context of bacterial nucleotide excision repair proteins, UvrABC endonuclease. Finally, the structures of the analogues were probed using chemical agents. The result was correlated with thermodynamic UV melting to draw light on the intriguing site selective formation of native C4-AP and DOB ICLs. Advisor: Prof. Marc M. GreenbergReaders: Prof. John P. Toscano Prof. Craig A. TownsendChair: Prof. John P. Toscano (jtoscano@jhu.edu)
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Investigating the Repair and Site Selective Formation of Interstrand Cross- Links Using Synthetic Analogues Derived from Oxidized Abasic Sites
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