Purification and characterization of nuclear factor III (origin recognition protein C), a sequence-specific DNA binding protein required for efficient initiation of adenovirus DNA replication

Nuclear factor III (NF-III, origin recognition protein C) is a cellular DNA binding protein that has high affinity for a DNA sequence contained within the adenovirus origin of DNA replication. We have purified NF-III more than 760-fold from HeLa nuclear extracts by a combination of conventional methods and DNA recognition site affinity chromatography. The NF-III polypeptide has an apparent molecular weight of 92,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sedimentation coefficient of the native protein is 3.1 S, and the Stokes radius is 61 Å. These values indicate that NF-III exists in solution as a somewhat asymmetric monomer. Purified NF-III binds specifically to a sequence within domain C of the adenovirus origin of replication and stimulates initiation of adenovirus DNA replication in in vitro reconstitution experiments. NF-III also binds specifically to a sequence element in the human histone H2B gene that is required for H2B-specific mRNA synthesis in vitro. Thus, NF-III may function as an activator of both viral DNA replication and cellular transcription.