Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells

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, β-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, β-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.

The DNA End-Binding Protein Ku Regulates Silencing at the InternalHMLandHMRLoci inSaccharomyces cerevisiae

“…Taken together with previous observations, our results suggest that Ku is endowed with two modes of binding to silent regions of DNA: it can bind telomeric regions directly via its DNA end-binding activity and it can bind HML and HMR as a result of protein-protein interactions. Previous observations have also implicated Ku in binding to internal chromosomal loci and suggest that Ku may play a role in the activation of transcription and possibly in initiation of replication (Barnes and Rio 1997;Ruiz et al 1999;Novac et al 2001;Walker et al 2001;Schild-Poulter et al 2003;Sibani et al 2005;Grote et al 2006;Shi et al 2007;Rampakakis et al 2008). The data presented here support and extend the data indicating that Ku binds to internal chromosomal loci and broaden our knowledge of the number of processes in which Ku plays a role at internal loci to include silencing.…”

Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication

“…In addition to its affinity for DNA ends and hairpins, Ku also binds to RNA (Yoo and Dynan, 1998;Tuteja and Tuteja, 2000), which could account for its strong molecular interaction with the hairpin-rich hY RNAs observed here. Ku has been shown to bind DNA replication origins in human cells and reduced levels of Ku result in cell growth defects and reduced rates of initiation of DNA replication (Sibani et al, 2005;Rampakakis et al, 2008). Therefore, Y RNAs and Ku might act in the same pathway regulating initiation of DNA replication in vertebrate somatic cells.…”

Ku is involved in cell growth, DNA replication and G1-S transition

“…On one hand, Ku80-haploinsufficient cells display decreased origin activation and delayed G1-S transition after synchronization in late G1 phase (Sibani et al, 2005b), suggesting a role of Ku80 in replication initiation. On the other hand, application of genotoxic stress to cells revealed a role of Ku in S-phase progression that seems to be different from its initiation role in the absence of cellular stress; Ku was implicated in the maintenance of the proliferating cell nuclear antigen (PCNA) on chromatin following ionizing radiation (IR) and chromosomal double-strand break (DSB) induction, and this role was independent of the DNA-PK kinase activity (Park et al, 2003).…”

“…PCR reactions were carried out in a total volume of 20 l, as previously described (Sibani et al, 2005b). The sequences and amplification conditions for all primer sets are listed in Table 1.…”