Oct-1 enhances the in vitro replication of a mammalian autonomously replicating DNA sequence.
A 186-base pair fragment of ors8, a mammalian autonomously replicating DNA sequence isolated by extrusion of nascent monkey DNA in early S phase, has previously been identified as the minimal sequence required for replication function in vitro and in vivo. This 186-base pair fragment contains, among other sequence characteristics, an imperfect consensus binding site for the ubiquitous transcription factor Oct-1. We have investigated the role of Oct-1 protein in the in vitro replication of this mammalian origin. Depletion of the endogenous Oct-1 protein, by inclusion of an oligonucleotide comprising the Oct-1 binding site,inhibited the in vitro replication of p186 to approximately 15-20% of the control, whereas a mutated Oct-1 and a nonspecific oligonucleotide had no effect. Furthermore, immunodepletion of the Oct-1 protein from the HeLa cell extracts by addition of an anti-POU antibody to the in vitro replication reactioninhibited p186 replication to 25% of control levels. This inhibition of replication could be partially reversed to 50-65% of control levels, a two- to threefold increase, upon the addition of exogenous Oct-1 POU domain protein. Site-directed mutagenesis of the octamer binding site in p186 resulted in a mutant clone, p186-MutOct, which abolished Oct-1 binding but was still able to replicate as efficiently as the wild-type p186. The results suggest that Oct-1 protein is an enhancing component in the in vitro replication of p186 but that its effect on replication is not caused through direct binding to the octamer motif.
Matheos
D D
DD
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Ruiz
M T
MT
Price
G B
GB
Zannis-Hadjopoulos
M
M
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D051863:Host Cell Factor C1; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology; D050811:Octamer Transcription Factor-1; D009841:Oligonucleotides / Q000378:metabolism / Q000494:pharmacology; D050809:POU Domain Factors; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C497692:HCFC1 protein, human; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D009841:Oligonucleotides; D050809:POU Domain Factors; C494165:POU2F1 protein, human; D010446:Peptide Fragments; D014157:Transcription Factors; D016590:Aphidicolin; D004247:DNA