Analysis of the DNA replication competence of thexrs-5 mutant cells defective in Ku86

Abstract

The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein,previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2,PCNA, DNA polymerase ϵ and δ, Primase and Topoisomerase IIα)were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oriβ) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oriβ origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells.

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

“…Further support for a role of Ku in the initiation of DNA replication (Novac et al, 2001;Matheos et al, 2002;Matheos et al, 2003) is provided by the observed decrease in origin usage, reflected by a significant 3.4-4.5-fold decrease in nascent strand DNA abundance in Ku80 +/-cells (Fig. 7).…”

Differentially Active Origins of DNA Replication in Tumor versus Normal Cells

“…The ability of the 20mer versions, found on human chromosome 19q13 and in the lamin B2 origin region, to confer autonomous replication activity when cloned into a plasmid was analyzed by the DpnI resistance assay, which is an indicator of semiconservative DNA replication, as previously described (36)(37)(38).…”

“…For transfections, HeLa cells were seeded in six-well plates at a density of 3 Â 10 4 per well, and f16 hours later were transfected with 3 Ag supercoiled plasmid DNA (2 Ag of each construct in Table 2 and 1 Ag pM1 SEAP), using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) as per instructions of the manufacturer. At 72 hours post-transfection, low molecular weight DNA was isolated and digested with DpnI, the DpnI-digested and undigested DNA were used to transform the DH5a strain of Escherichia coli and the relative in vivo DNA replication of each transfected plasmid was determined by counting the number of colonies in a bacterial retransformation assay, as previously described (36)(37)(38). The levels of secreted human placental alkaline phosphatase, determined by the SEAP Reporter Gene Assay kit (Roche Molecular Biochemicals), as per specifications of the manufacturer, were used to normalize the transfection efficiency.…”

The Ku Complex in Silencing the Cryptic Mating-Type Loci ofSaccharomyces cerevisiae

“…Instead, Ku and Sir4 may be relying on some other silencer-recognition mechanism. In this regard, it is intriguing that the effects of yeast Ku on ORC binding in vitro have been reported (Shakibai et al 1996) and that in mammalian cells Ku associates with replication origins and has been found in a complex with known replication proteins, including ORC2 (Novac et al 2001;Matheos et al 2002Matheos et al , 2003. In addition, recent two-hybrid analyses of Orc subunits report an interaction between Orc2 and Sir4 (Matsuda et al 2007).…”