Cofractionation of HeLa cell replication proteins with ors-binding activity.
Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases alpha and delta, topoisomerase II, and replication protein A, (RP-A).
Ruiz
M T
MT
McGill Cancer Centre, Department of Medicine, McGill University, Montréal, Québec, Canada.
Pearson
C E
CE
Nielsen
T
T
Price
G B
GB
Zannis-Hadjopoulos
M
M
D001483:Base Sequence; D002458:Cell Fractionation; D004257:DNA Polymerase II / Q000302:isolation & purification*; D004258:DNA Polymerase III; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000378:metabolism; D011485:Protein Binding; D051716:Replication Protein A
D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D004250:DNA Topoisomerases, Type II