RIP60, a Mammalian Origin-Binding Protein, Enhances DNA Bending near the Dihydrofolate Reductase Origin of Replication

Abstract

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeTR fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddie, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)_3-4 tracts, designated bend elements Bl to B5, that are spaced 10 bp apart. DNA bending directed by elements Bl to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4°C. Bend element B5, which is in inverse orientation relative to elements Bl to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.