Studies on origin binding proteins involved in mammalian DNA replication

The objective of this thesis is to investigate the proteins interacting with specific DNA sequences, termed origins of DNA replication in vivo. Two previously described origin binding proteins, OBA/Ku and CBP/14-3-3 were analyzed. Previously, Ku was shown to bind to A3/4, a 36-bp origin sequence, in vitro, and 14-3-3 isoforms were identified as cruciform binding proteins (CBP) which interact with cruciform structures present in mammalian replication origins.;Here, the in vivo association of Ku and 14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey (CV-1) replication origins ors8 and ors12, by formaldehyde cross-linking, followed by chromatin immunoprecipitation (Chip) and quantitative PCR analysis. The involvement of 14-3-3 in mammalian DNA replication was also analyzed by studying the effect of anti-14-3-3beta, epsilon, gamma, and zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8.;Ku and 14-3-3beta, epsilon, gamma, zeta and sigma isoforms were found to be associated with mammalian origins of DNA replication and their association was the highest in cells synchronized at the G1/S phase of the cell cycle. In addition, Anti-14-3-3epsilon, gamma and zeta antibodies inhibited p186 replication by approximately 30--80%.;The Ku80 mutant (xrs-5) and deficient (Ku80-/- MEFs) cell lines were also tested for their ability to replicate p186, in vitro. Whole cell (WCE) and cytoplasmic cell extracts from the xrs-5 cells replicated p186 with the same efficiency as are wild-type (wt) CHO K1 cells. In contrast, xrs-5 nuclear extracts did not possess any detectable replication activity, while the Ku80 -/- WCE had a decrease of ∼70% in their ability to support p186 replication, by comparison to the wt Ku80-/- extracts. Furthermore, in vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% by comparison to CHO K1 cells.;The in vivo association of Ku with the Chinese hamster DHFR oribeta or the mouse Adenosine deaminase (ADA) origins of DNA replication was examined in both the Ku80 mutant (xrs-5) and deficient (Ku80-/-) cell lines, and in their respective wild-type counterparts. Anti-Ku antibodies failed to immunoprecipitate a detectable amount of Ku from the either xrs-5 or Ku80-/- cells in the origin-containing-sequence, in contrast to the wild type cells, wherein Ku was found to be associated with the oribeta and ADA origins, respectively.;The data implicate Ku antigen in DNA replication and suggest the existence of another protein in rodent cells that is able to substitute for Ku function. They also indicate a novel function for Ku and the 14-3-3 isoforms beta, epsilon, gamma, zeta and sigma, as origin-binding-proteins in vivo, which provides a better understanding of the chromosomal association and DNA binding sequences of mammalian initiator proteins with origins of replication in their natural chromosomal environment.