In vivo association of Ku with mammalian origins of DNA replication

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 as sociation of Ku with mammalian origins of DNA replication was analyzed by s tudying its association with ors8 and ors12, as assayed by formaldehyde cro ss-linking, followed by immunoprecipitation and quantitative polymerase cha in reaction analysis. The association of Ku with ors8 and ors12 was also an alyzed as a function of the cell cycle. This association was found to be ap proximately fivefold higher in cells synchronized at the G1/S border, in co mparison with cells at G0, and it decreased by approximately twofold upon e ntry of the cells into S phase, and to near background levels in cells at G 2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibr oblasts. A decrease of similar to 70% in in vitro DNA replication was obser ved when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extr acts. The results indicate a novel function for Ku as an origin binding pro tein, which acts at the initiation step of DNA replication and dissociates after origin firing.