Small molecule-based approaches toward elucidation of structure and properties of abasic site-derived interstrand DNA crosslinks
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Damage to DNA is both a naturally occurring and environmentally induced process that can have serious biological consequences, such as mutation and cancer. The interstrand crosslink is a particularly harmful form of DNA damage that interferes with important processes within a cell. Additionally, these lesions present a challenge to normal cellular DNA repair mechanisms, which can enhance their damaging effects. For these reasons, many chemotherapeutic drugs are specifically designed to form interstrand crosslinks in cancer cells, which can have the desired effect of tumor suppression. Unfortunately, these drugs are often toxic to normal cells as well, causing a variety of serious side effects associated with chemotherapy. Our lab has recently identified a type of interstrand crosslink that is formed not by the action of a drug, but rather by the reaction of DNA with itself. More specifically, we have found that DNA bases adenine and guanine can react with naturally occurring abasic sites within DNA to form a chemical linkage between the two strands of a DNA double helix, i.e., an interstrand crosslink. We suspected that this particular DNA lesion might represent a significant type of natural DNA damage, and therefore, our lab set out to investigate some specific properties of this lesion in order to make inferences about its potential biological outcomes. We employed the techniques of NMR spectroscopy and mass spectrometry to evaluate the chemical structure of the interstrand crosslink formed between guanine bases and abasic sites (dG-Ap crosslinks) in DNA. Our findings indicate that the structure of these lesions gives them substantial stability, which was confirmed by monitoring their time-dependent decomposition. The stability of these crosslinks, as demonstrated by our experiments, suggests that their persistence in a biological context may enhance their ability to disrupt critically important cellular functions, such as DNA transcription and replication. We also wanted to investigate the chemical bases that explain why these types of crosslinks form. This was accomplished by monitoring crosslink formation within a DNA molecule as well as outside the DNA context by using small molecules that resemble the reacting DNA constituents as "stand-ins". Because these small molecule stand-ins can be modified to examine the effect of a variety of chemical properties on a specific chemical reaction, we can measure how these properties affect the crosslink formation reaction within a DNA molecule by direct comparison to trends observed in the analogous small molecule reactions. We used these methods to assess the abilities of the four DNA bases (A, C, G and T) to form interstrand crosslinks with abasic sites as well as speculate as to the specific chemical properties that drive crosslink formation. Our results showed that, while fundamental chemical properties can be used to explain differences in crosslinking ability to some extent, the most determinant factor affecting crosslink formation within a DNA duplex may be the local structure of the DNA molecule itself. Together, these results provide further insight into the structure and properties of abasic site-derived interstrand DNA crosslinks that will inform ongoing investigations of these lesions. Until present, all of our studies involving DNA have been conducted using a synthetic form of the DNA molecule, rather than DNA from living organisms. The work described here may be useful in the development of methods for detection of these crosslinks in genomic DNA.