UC San Diego

The maintenance of the genome is one of the foremost functions of all living organisms. The genome undergoes constant assault by environmental hazards, leaving behind different types of DNA alterations. Unrepaired DNA damage has been linked to several neurological diseases. Among many proteins involved in DNA damage repair, polynucleotide kinase 3' phosphatase (PNKP) is a key regulator of DNA repair. PNKP removes 3'-phosphate at the DNA breaks. Our collaborator has reported that PNKP levels are unchanged in poly-glutamine neurological diseases, it is inactive. Another kinase phosphatase, phosphofructokinase fructose-2,6-bisphosphatase 3 (PFKFB3), is linked to DNA repair. My thesis addresses the question if the product of PFKFB3 supports PNKP activity. Here I document that PNKP interacts with fructose-2,6-bisphosphate produced by PFKFB3. Furthermore, I describe the enzymatic production and purification of fructose-2,6-bisphosphate in bulk quantities. PNKP 3’ Phosphatase activity levels are significantly lower in the nuclear extracts of HD and SCA3 patients’ brains. Exogenous F2,6BP rescued PNKP 3’ phosphatase activity in the brain nuclear extracts of those samples. Moreover, F2,6BP association with PNKP was indicated by bio-layer interferometry and protein stability assays. We thus postulate that F2,6BP serves in vivo as a cofactor for the proper 3’ phosphatase of PNKP and, brain health.