Abstract
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.
Highlights
DNA replication is regulated at the level of initiation, and at the level of pre-replication complex formation at replication origins
Several replication origins have been mapped to specific DNA sequences, indicating that the process of initiation of DNA replication does not occur randomly throughout the genome (Huberman and Riggs, 1968; Hand, 1978; Goldman et al, 1984; Pierron et al, 1984; Jalouzot et al, 1985; Gilbert, 1986; Zannis-Hadjopoulos and Price, 1998; Zannis-Hadjopoulos and Price, 1999), to date, no human DNA initiator protein has been identified that binds to replication origins in a sequence-specific manner (Dhar et al, 2001a; Bell and Dutta, 2002)
We examined the role of Ku in the initiation of DNA replication, using HCT116 Ku80+/– haploinsufficient cells
Summary
DNA replication is regulated at the level of initiation, and at the level of pre-replication complex (pre-RC) formation at replication origins (reviewed by Dutta and Bell, 1997; Bell and Dutta, 2002; Sharova and Abramova, 2002; Li et al, 2003). In vitro analyses have shown that the ORC complex alone does not possess sequence-specific DNA binding activity (Dhar et al, 2001a; Bell and Dutta, 2002), with the exceptions of those of Schizosaccharomyces pombe and Saccharomyces cerevisiae. In S. pombe, the AT hook of the Orc subunit of the complex allows sequence-specific binding to AT rich regions (Chuang and Kelly, 1999; Moon et al, 1999), whereas in S. cerevisiae, ATP-bound Orc1p is responsible for ScORC attachment to origins (Klemm et al, 1997). It was postulated that ORC might interact with a sequence-specific binding protein that recruits ORC to the origins (Bell and Dutta, 2002; ZannisHadjopoulos et al, 2004)
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