Rep interaction with cellular proteins. Cells which over-express the larger Rep polypeptides have a distinctive phenotype. These cells appear to be blocked in the cell cycle and are unable to undergo mitosis. Our results demonstrate that Rep 52 binds to and inhibits the cyclic AMP responsive cellular kinases, PrKX and PKA. The Rep 68 derived polypeptide bound very weakly to PrKX with minimal inhibitory effect over the concentration range of Rep 68 examined in these assays. A corresponding inhibition of PrKX autophosphorylation by Rep 52 but not Rep 68 was observed. No interactions between Rep 68 and PKA were detected and only very low levels of phosphorylation inhibition activity was detected in the in vitro assays. The cyclic AMP (cAMP) activated protein kinase A, PKA, responds to intracellular increases of cAMP. PKA is a primary responder to intracellular cAMP concentrations which fluctuate with the cell cycle with peak levels observed during G1 and G2/M phases. One target of PKA phosphorylation is the transcription factor modifying protein, CREB. Phosphorylated CREB interacts with CBP, a transcription activator, which can activate transcription via binding to cAMP binding protein responsive elements. Examples of genes induced by CREB include: c-jun, c-myb, myoD, E2F-1, YY1, cyclin A, and PCNA. Helicase activities of the p19 Rep proteins. The two Rep polypeptides derived from transcription initiated at p19, Rep 52 and Rep 40, lack the amino terminus 224 residues of Rep 68 and Rep 78. Sequence specific DNA binding and DNA endonuclease activities map to the amino terminus. DNA helicase and ATPase activities reside in the carboxy terminus that is common to all four Rep polypeptides. We have demonstrated that Rep 52 possesses DNA helicase activity. The level of Rep 52 and Rep 78 helicase activities were similar as estimated by the extend of unwinding per unit of Rep protein. The polarity of Rep 52 unwinding was as 3' to 5'. These results indicate that Rep 52 functions as a monomeric helicase that lacks sequence specificity. Our data demonstrate that all AAV2 Rep proteins have enzymatic activity and at least two of these activities are common to the p5 and p19 Rep proteins.
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