In Vivo Association of Ku with Mammalian Origins of DNA Replication

Abstract

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association withors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80+/+ and Ku80−/− mouse embryonic fibroblasts. A decrease of ∼70% in in vitro DNA replication was observed when the Ku80−/− extracts were used, compared with the Ku80+/+ extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.