1075. Cellular Localization and Cell Cycle Dynamics of φC31 Integrase in Mammalian Cells

Abstract

Top of pageAbstract Bacteriophage |[phiv]|C31 integrase is able to catalyze a unidirectional, site-specific DNA recombination reaction. Constrained target sequence preference, combined with an ability to function in a eukaryotic environment, have made |[phiv]|C31 integrase an important tool in higher organism genetic manipulation, including gene therapy applications. To better understand the properties of the enzyme in a mammalian cellular context, we constructed a plasmid expressing |[phiv]|C31 integrase with a hemagglutin (HA) epitope tag. Using immunoflurescence, we determined that |[phiv]|C31 integrase localized to the cytoplasm in HeLa cells. Intriguingly, we found that while |[phiv]|C31 integrase localization did not change during cell cycle progression, the absolute abundance of the enzyme declined rapidly after passage through the G1/S checkpoint. We have shown that this effect is due to degradation by the 26S proteasome. In cells treated with a chemical proteasome inhibitor, we found that |[phiv]|C31 integrase appeared to localize to a peri-centriolar inclusion body, as determined by co-staining of the centrosomal marker |[gamma]|-tubulin. Specialized centrosome-associated proteasomal complexes, known as 'aggresomes', have been identified as sites for clearance of misfolded or aggregated protein products. Our results suggest that, when expressed in cycling mammalian cells, |[phiv]|C31 integrase may be actively trafficked to aggresomes and degraded. The implications of these results for gene therapy and avenues to use this information for enhancement of the integration reaction will be presented.