Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells.
One of the functions of the abundant heterodimeric nuclear protein, Ku (Ku70/Ku80), is its involvement in the initiation of DNA replication through its ability to bind to chromosomal replication origins in a sequence-specific and cell cycle dependent manner. Here, using HCT116 Ku80+/- cells, the effect of Ku80 deficiency on cell cycle progression and origin activation was examined. Western blot analyses revealed a 75% and 36% decrease in the nuclear expression of Ku80 and Ku70, respectively. This was concomitant with a 33% and 40% decrease in chromatin binding of both proteins, respectively. Cell cycle analysis of asynchronous and late G1 synchronized Ku80+/- cells revealed a prolonged G1 phase. Furthermore, these Ku-deficient cells had a 4.5-, 3.4- and 4.3-fold decrease in nascent strand DNA abundance at the lamin B2, beta-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, beta-globin and c-myc origins was decreased by 1.5-, 2.3- and 2.5-fold, respectively, whereas that of Ku70 was similarly decreased (by 2.1-, 1.5- and 1.7-fold, respectively) in Ku80+/- cells. The results indicate that a deficiency of Ku80 resulted in a prolonged G1 phase, as well as decreased Ku binding to and activation of origins of DNA replication.
Sibani
Sahar
S
McGill Cancer Center, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Price
Gerald B
GB
Zannis-Hadjopoulos
Maria
M
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase; D000072200:Ku Autoantigen; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding; D013696:Temperature
D034961:Antigens, Nuclear; D002843:Chromatin; D004268:DNA-Binding Proteins; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen