| A 186-bp fragment of ors8, a mammalian DNA replication origin, was previously identified as the minimal sequence required for origin function in vivo and in vitro. The 186-bp origin contains, among other features, an octamer motif, serving as the binding site for the transcription factor Oct-1, and an A3/4-homologous region, serving as the binding site for the origin binding protein, OBA. The research objective of this thesis is to investigate the role of Oct-1 and OBA in mammalian in vitro DNA replication.;Depletion of the Oct-1 protein inhibited in vitro DNA replication to basal levels. The inhibition was partially reversed upon the exogenous addition of Oct-1 POU protein. Site-directed mutagenesis of the octamer motif in the origin resulted in a mutant clone that lacked Oct-1 binding but replicated efficiently. The results suggest that Oct-1 is an enhancing component in DNA replication but its effect is not caused through direct DNA binding, but rather through protein-protein interactions.;OBA was purified from HeLa cells through its ability to interact with the 186-bp origin. OBA binds to A3/4, a 36-bp mammalian origin sequence. The DNA binding subunit of OBA is identical to the 86 kDa subunit of Ku antigen. Depletion of OBA/Ku, by inclusion of anti-Ku antibodies to the in vitro replication reaction of p186, inhibited DNA replication. Furthermore, addition of the A3/4 oligonucleotide to the replication reaction of mammalian origin-containing plasmids (p186, pors12, and pX24) inhibited replication. This inhibition was reversed upon addition of OBA. In contrast, SV40 in vitro replication remained unaffected. OBA also possessed DNA helicase activity. By co-immunoprecipitation analyses, OBA was found to associate with DNA polymerases delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, and Oct-1.;The replication activity of xrs-5, a derivative of Chinese hamster ovary (CHO) K1 cell line defective in Ku86, was also examined. Total and cytoplasmic extracts from xrs-5 cells replicated p186 DNA as efficiently as CHO K1 extracts. In contrast, xrs-5 nuclear cell extracts lacked DNA replication activity, which was restored upon addition of OBA.;The data implicate OBA/Ku in a direct role in the initiation of mammalian DNA replication as an origin-specific binding protein with helicase activity. The physical association of OBA/Ku with Oct-1 and the replication machinery reveals a possible mechanism by which these proteins are recruited to DNA replication origins. A cytoplasmic Ku-like origin-specific binding protein likely compensates for the absence of the endogenous OBA/Ku in xrs -5 cells.;These studies provide a better understanding of the proteins that bind to mammalian origins, thereby aiding in understanding the mechanisms that regulate the initiation of DNA replication. |
