Biophysical and biochemical characterisation of DNA-based inhibitors of the cytosine-mutating APOBEC3 enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

With the rise of antiviral and anticancer drug resistance, a new approach must be taken to overcome this burden. The APOBEC3 (A3) family of cytosine deaminases hypermutate cytosines to uracils in single-stranded DNA (ssDNA). These enzymes act as double-edged swords: on one side they protect humans against a range of retroviruses and other pathogens, but several A3s are exploited by viruses and cancer cells to increase their rate of evolution using the enzyme’s mutagenic actions. This latter mode permits escape of cancer cells from the adaptive immune response and leads to the development of drug resistance. In particular, APOBEC3B (A3B) is considered to be a main driving source of genomic mutations in cancer cells. Inhibition of A3B, while retaining the beneficial actions of the other A3 in the immune system, may be used to augment existing anticancer therapies. In this study, we showed for the first time that short ssDNAs containing cytosine analogue nucleosides, 2'-deoxyzebularine (dZ) or 5-fluoro-2'-deoxyzebularine (5FdZ) in place of the substrate 2'-deoxycytidine (dC) in the preferred 5'-TC motif, inhibit the catalytic activity of A3B. However, as most A3 enzymes (except A3G) prefer to deaminate ssDNA with a 5'-TC motif, selective A3B inhibition was uncertain. We noted that nucleotides adjacent to the 5'-CCC motif influence the dC deamination preference of A3A, A3B, and A3G’s. Replacement of the A3B’s preferred dC in the 5'-CCC motif with dZ (5'-dZCC) led to the first selective inhibitor of A3B, in preference to A3A and A3G. Furthermore, using small-angle X-ray scattering (SAXS) we obtained the first model of a full-length two-domain A3 in complex with a dZ-ssDNA inhibitor. Our model showed that the ssDNA was largely bound to the C-terminal domain (CTD) with limited contact to the N-terminal domain in solution, due to the high affinity of the dZ for the CTD active-site. Our work provides a new platform for use of ssDNA-based inhibitors in targeting the mutagenic action of the A3B. Further developments using more potent inhibitors will help to achieve inhibition in cellular studies, with the ultimate goal to complement anti-cancer and antiviral treatments.