The APOBEC3 (A3) family of enzymes catalyze the deamination of cytosine to uracil in single stranded (ss)DNA, and studies have shown that APOBEC3A (A3A) and APOBEC3B (A3B) activity are sources of mutation that promote drug resistance and tumor evolution in cancer patients. While studies have shown that the deaminase activity of these proteins promotes poor clinical outcomes, it is poorly understood whether these mutations are occurring via DNA or RNA editing. Though A3s have been shown to bind RNA, A3A and A3B selectively deaminate ssDNA over RNA. The discrete molecular features that dictate ssDNA binding and A3-mediated deamination over RNA binding are not well characterized on a molecular level. Using molecular dynamics simulations our studies suggest that the selective deamination is due to the difference in the sugar conformation between the ribose of RNA and 2¢-deoxyribose of ssDNA. Although structurally similar, the hydroxyl present at the 2¢-position of ribose results in a 3¢-endo conformation of the sugar, whereas the geminal hydrogens on the 2¢-deoxyribose promote a preferred 2¢-endo conformation. These differences in sugar conformation may disrupt key interactions between the substrate and protein that confer the molecular recognition properties of A3-substrate binding. To experimentally validate our computational findings, we have assessed both the binding affinity and substrate turnover of oligonucleotides containing cytidine analogues that prefer either the 2¢-endo (DNA) or 3¢-endo (RNA) conformations. Using fluorescence polarization and differential scanning fluorimetry to assess oligonucleotide binding, and gel-based deamination assays to query substrate deamination, we provide critical insights into A3-substrate recognition in this poster presentation.

Characterization of the effect of sugar conformation on APOBEC3 substrate recognition

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ACS Fall 2022

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