Human WRNIP1, a Werner DNA helicase interacting protein 1, was expressed in insect cells and E. coli . The purified protein behaved as a homo-oligomeric complex with a native molecular mass indicative of an octamer, and the complex copurified with an ATPase activity that was stimulated by double-stranded DNA ends. As suggested by genetic studies of budding yeast WRNIP1/Mgs1, the purified human WRNIP1 complex interacted physically with human DNA polymerase δ δ δ δ ( pol δ δ δ δ ), stimulating its DNA synthesis activity more than fivefold in the presence or absence of proliferating cell nuclear antigen. Analysis of reaction products demonstrated the stimulation to be partly due to an increased processivity of pol δ δ δ δ but more importantly to an increase in its initiation frequency. Addition of ATP to reactions partially suppressed stimulation by WRNIP1. Furthermore, a mutant WRNIP1 lacking ATPase activity could stimulate pol δ δ δ δ normally but was insensitive to suppression by ATP. These results indicate that WRNIP1 functions as a modulator for initiation or restart events during pol δ δ δ δ -mediated DNA synthesis and that its ATPase activity is utilized to sense DNA ends and to regulate the extent of stimulation.

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Pim-1, an oncogene product of serine/threonine kinase, has been found to play roles in apoptosis induction/ suppression, cell-cycle progression and transcriptional regulation by phosphorylating the target proteins involved in these processes. The target proteins phosphorylated by Pim-1, including p100, Cdc25A, PAP-1 and heterochromatin protein 1, have been identified. The precise functions of Pim-1, however, are still poorly understood. In this study, we identified tumor necrosis factor receptor-associated factor 4-associated factor 2/sorting nexin 6 (TFAF2/SNX6) as a Pim-1-binding protein, and we found that TFAF2/SNX6 was phosphorylated and translocated from the cytoplasm to nucleus by Pim-1. This translocation of the protein was not affected by Pim-1-dependent phosphorylation. Since sorting nexins, including TFAF2/SNX6, have been reported to be located in the cytoplasm or membrane by association with several receptors of tyrosine-or serine/ threonine-kinase, this is the first report of TFAF2/SNX6 being located in the nucleus after binding to Pim-1. ß

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