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 A. 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.
O'Neill
E A
EA
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Kelly
T J
TJ
D000256:Adenoviridae / Q000235:genetics*; D002846:Chromatography, Affinity; D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D051863:Host Cell Factor C1; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050811:Octamer Transcription Factor-1; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D051863:Host Cell Factor C1; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D014157:Transcription Factors; D004262:DNA Restriction Enzymes