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Oct‐1 enhances the in vitro replication of a mammalian autonomously replicating DNA sequence
Author(s) -
Matheos Diamanto D.,
Ruiz Marcia T.,
Price Gerald B.,
ZannisHadjopoulos Maria
Publication year - 1998
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(19980301)68:3<309::aid-jcb3>3.0.co;2-y
Subject(s) - ter protein , biology , replication factor c , pou domain , histone octamer , origin recognition complex , dna replication , microbiology and biotechnology , dna replication factor cdt1 , control of chromosome duplication , licensing factor , origin of replication , eukaryotic dna replication , seqa protein domain , s phase , oligonucleotide , replication protein a , dna , dna binding protein , genetics , transcription factor , gene , histone , nucleosome , homeobox
A 186‐base pair fragment of ors 8, 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. J. Cell. Biochem. 68:309–327, 1998. © 1998 Wiley‐Liss, Inc.