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 p hase, has previously been identified as the minimal sequence required for replication function in vitro and in vivo. This 186-base pair frag ment contains, among other sequence characteristics, an imperfect cons ensus binding site for the ubiquitous transcription factor Oct-1. We h ave 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 addit ion of exogenous Oct-1 POU domain protein. Site-directed mutagenesis o f the octamer binding site in p186 resulted in a mutant clone, p186-Mu tOct, which abolished Oct-1 binding but was still able to replicate as efficiently as the wild-type p186. The results suggest that Oct-1 pro tein is an enhancing component in the in vitro replication of p186 but that its effect on replication is not caused through direct binding t o the octamer motif. (C) 1998 Wiley-Liss, Inc.