We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.

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BackgroundEpstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied.Methods and FindingsUsing Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate.ConclusionsOur data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.

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