Delineation of the domains required for physical and functional interaction of p14ARF with human topoisomerase I.

We recently reported an interaction between the p14(ARF) protein and human topoisomerase I (Topo I) resulting in the stimulation of the relaxation activity of Topo I. Our data showed that the complex between the two proteins was located within the nucleolus. In the present work, we have investigated the regions of p14(ARF) involved in this interaction by using targeted point mutagenesis and deletion mutants. A region encompassing exon 2-encoded sequence was required for physical binding of p14(ARF) to Topo I as well as for stimulatory activity of the enzyme. Exon 1 beta-encoded segment was not implicated in the interaction. Moreover, among p14(ARF) point mutants selected for their high conservation among different mammalian species, mutant p14(ARF) (RR87, 88AA) did not stimulate Topo I in spite of its association with the enzyme, suggesting its direct implication in the functional activity of ARF. In contrast, one mutant, p14(ARF) (R71A), was more efficient than wild-type protein to activate Topo I, suggesting that this residue is a key element to modulate Topo I activity. Finally, only ARF-Topo I complexes containing p14(ARF) exon 2 segment were found to be localized in the nucleolus, suggesting that this subnuclear location is linked to the biological function of the ARF-Topo I complex.