COFRACTIONATION OF HELA-CELL REPLICATION PROTEINS WITH ORS-BINDING ACTIVITY

Ors (origin enriched sequence) 8 is a mammalian autonomously replicati ng 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 identifi ed as the minimal sequence required for origin function, since upon it s deletion the in vivo and in vitro replication activity of this ors i s abolished. We have fractionated total HeLa cell extracts on a DEAE-S ephadex 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 specif ic manner. The same fraction is able to support the in vitro replicati on of ors 8 plasmid. The ors binding activity (OBA) present in this fr action 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 weig hts 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). (C) 1995 Wiley-Liss, Inc.