DNA replication initiates at origins with the origin recognition complex (ORC) binding and activating initiation. Although ORC specifically binds origins in vivo, it possesses no sequence specific DNA binding activity in vitro. Thus, it is thought to interact with another protein that recruits it to the origin. The heterodimeric Ku (Ku70/Ku80) protein is one candidate. Ku is an abundant nuclear protein that has recently been implicated in the initiation of DNA replication, but the mechanism by which it performs this function is yet unknown. Here, using haploinsufficient human HCT116 Ku80 +/- cells that express 25-40% Ku80 protein, the origin activity and origin-association of other replication proteins were examined in a Ku80-deficient environment. Ku80 was not found to influence the expression of replication proteins, except for Ku70, its partner, which displayed a reduction of its protein expression to 64% of wildtype cells. Ku80+/- cells displayed a prolongation of G1 phase by 2 hours, but normal progression through S-phase. Furthermore, the activity of the early-activated replication origins of the lamin B2, beta-globin and c-myc loci decreased by 4.5-, 3.4-, and 4.3-fold, respectively. This was associated with a 2.1-, 1.5- and 1.7-fold decrease in the association of Ku80 with these origins, respectively, and a 1.5-, 2.3- and 2.5-fold decrease in that of Ku70, respectively. Moreover, the origin-association of three of the six subunits of the ORC complex, Orc-3, -4, and -6 was significantly decreased in Ku80 +/- cells, indicating decreased ORC complex formation at the origins. In contrast, the association of Orc-2 was not influenced by Ku80 deficiency, nor was the association of Ku80 with origins affected by Orc2 deficiency. Overall, these results suggest that Ku binds origins to allow assembly of the ORC complex, which is necessary for origin licensing and activation. Ku80-/- murine embryonic fibroblasts (MEFs) are viable but display a prolonged doubling time of 43 hours vs. 23 hours for wildtype cells. To analyze the effect of Ku80 absence on DNA replication, the late-firing murine adenosine deaminase origin was fine-mapped by nascent strand abundance analysis. Two origins were identified, mAdA-1 and mAdA-C, with 34- and 29-fold greater nascent strand abundance relative to non-origin DNA. These origins were able to replicate episomally when transiently transfected into MEF cells. In Ku80-/- cells, in situ and episomal DNA replication assay of both origins indicated that Ku80 did not influence their DNA replication activity.

This item was digitized as part of a project to share McGill's intellectual legacy with the public. If you are the copyright holder or a relative of the copyright holder who is deceased, you may request withdrawal by emailing escholarship.library@mcgill.ca. The full policy for eScholarship may be accessed here: https://www.mcgill.ca/libraries/research-services/escholarship/policy