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First published online 14 November 2002
doi: 10.1242/jcs.00156

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

Diamanto Matheos1,2, Olivia Novac1,2, Gerald B. Price1 and Maria Zannis-Hadjopoulos1,2,*

1 McGill Cancer Centre, McGill University, Montréal, Québec, Canada, H3G 1Y6
2 Department of Biochemistry, McGill University, Montréal, Québec, Canada, H3G 1Y6



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  		Fig. 5. Quantification of DNA abundance in the DHFR oriß region by real-time PCR. (A) Map of the DHFR initiation zone, containing the ß, ß' and {gamma} initiation sites and encompassing the DHFR and 2BE2121 genes. The arrows represent the location of the amplification product using a primer set within the oriß region. (B) Quantitative realtime PCR using the LightCycler instrument, using a primer set within the oriß region or ~17 kb downstream from the DHFR gene (AF028017), with DNA template extracted from the immunoprecipitation with anti-Ku70, anti-Ku86, anti-clone 162 antibodies or NGS from crosslinked or untreated CHO K1 or xrs-5 cells. Each bar represents two experiments, and one standard deviation is indicated.

 


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  		Fig. 4. Ku is associated with the DHFR oriß origin of DNA replication in CHO K1 cells but not in xrs-5 mutant cells. (A) Standard curve, using CHO K1 genomic DNA as template, used for the quantification of DNA abundance in the origin-containing sequence (oriß) or in the non-origin-containing sequence (17 kb downstream from the DHFR gene amplified by AF028017 primer set) by real-time PCR. The LightCycler software 3.5 calculates the copy number of molecules, amplified by the respective primer set, by plotting on the x-axis the logarithm of fluorescence and on the y-axis the cycle number and setting a baseline x-axis. (B) Melting peak analysis of the 152 bp or 255 bp PCR amplification products performed at the end of the PCR amplification cycle. Melting peaks were generated by plotting the negative derivative of the SYBR Green fluorescence with respect to temperature (—dF/dT) against temperature (°C). (C) LightCycler PCR amplification products, amplified with the oriß primer set, were separated on a 2% agarose gel, visualized with ethidium bromide and photographed with an Eagle Eye apparatus. Lane 1 represents a 50 bp marker ladder (Amersham). Template DNA was as follows. Lane 2, 1/20th of DNA recovered from immunoprecipitation with normal goat serum (NGS) in crosslinked CHO K1 cells. Lanes 3-6, CHO K1 genomic DNA (S1-S4) from untreated cells used to build the standard curve in A. Lanes 7-14, 1/20th of DNA recovered from immunoprecipitation with anti-Ku70, anti-Ku86 or anti-clone162 from CHO K1 or xrs-5 cells crosslinked or untreated with formaldehyde. (D) As for C but with the AF028017 primer set. Template DNA was as follows. Lane 1 represents a 50 bp marker ladder (Amersham). Lanes 2-4, CHO K1 genomic DNA (S1-S3) from untreated cells used to build the standard curve in (A). Lane 5, water was used as template. Lanes 6-8, 1/20th of DNA recovered from immunoprecipitation with anti-Ku70 anti-Ku86, or anti-clone162 from CHO K1 cross-linked cells.

 


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  		Fig. 1. In vivo DNA replication activity of CHO K1 and xrs-5 cells. CHO K1 or xrs-5 cells were transfected with either p186 or pBR322 DNA. 72 hours post-transfection, plasmid DNA was isolated by the method of Hirt, then purified and digested with DpnI. The DpnI-digested DNA was then used to transform E. coli. The number of bacterial colonies produced was counted, corrected for the amount of DNA recovered and related to the positive control reaction with the CHO K1 cells, which was taken as 100%. The bars represent the error from the average of two experiments performed in triplicate.

 


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  		Fig. 2. Immunoblot analysis of CHO K1 and xrs-5 cell extracts. Nuclear (N; panels A and B, lanes 3 and 5) or cytoplasmic (C; panels A and B, lanes 2 and 4) cell extracts from CHO K1 or xrs-5 cells were examined by western blotting using a Ku86 (A) or Ku70 (B) antibody. HeLa whole-cell extracts (lane 1) were used as positive controls.

 


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  		Fig. 3. Immunoblot analysis of replication proteins in CHO K1 versus xrs-5 cells. Nuclear (N; panels A-F) cell extracts from CHO K1 or xrs-5 cells were examined by western blotting using anti-ORC2 (A), anti-PCNA (B), anti-DNA Polymerase {epsilon} (C), anti-Polymerase {delta} (D), anti-Primase (E) and anti-Topoisomerase II{alpha} (F) antibodies.

 


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  		Fig. 6. A3/4 binding activity in CHO K1 and xrs-5 cell extracts. (A) Nuclear (N) cell extracts from CHO K1 or xrs-5 cells were mixed with a radiolabeled double-stranded A3/4 DNA probe. HeLa nuclear (N) and cytoplasmic (C) extracts were used as a positive control (HeLa NC). Following the binding reaction, clone 162 or control antibody was added to the mixture, as indicated. The DNA-protein complexes were separated by 6% PAGE. The Ku70/Ku86-A3/4 (*), Ku70/Ku69-A3/4 (**) and the supershifted complexes (***) are indicated. (B) As in panel A except that cytoplasmic (C) cell extracts from CHO K1 and xrs-5 cells were used.

 


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  		Fig. 7. Competition/bandshift assay using CKO K1 and xrs-5 cytoplasmic cell extracts. The electrophoretic mobility shift assay was performed by adding cytoplasmic (C) cell extracts from xrs-5 (lanes 2-6) or CHO K1 (lanes 7-11) to a mixture containing radiolabeled A3/4 oligonucleotide (lane 1). Some reactions contained no additional competitor DNA (lanes 2 and 7). Lanes 3 and 8 contained 50x molar excess of cold A3/4 competitor, lanes 4 and 9 contained 500x cold A3/4 competitor, lanes 5 and 10 contained 50x non-specific cold competitor and lanes 6 and 11 contained 500x non-specific competitor. The positions of the protein-DNA complexes and of the free probe are indicated.

 


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  		Fig. 8. In vitro replication activity of the CHO K1 and xrs-5 cell extracts. (A) Typical autoradiograph of DNA replication products. p186 was incubated in reaction mixtures containing CHO K1 or xrs-5 total cell extracts. The DNA was purified, concentrated and a sample was digested with 1 unit of DpnI for 1 hour at 37°C. The DpnI-digested (lanes 1-12) samples were subjected to electrophoresis on 1% agarose gel. The supercoiled (I), relaxed circular (II) and linear (III) forms of the plasmid and the DpnI-digestion products are indicated. Duplicate samples are shown. (B) xrs-5 nuclear cell extracts do not replicate p186. In vitro DNA replication assays were performed with CHO K1 or xrs-5 nuclear and cytoplasmic (NC), cytoplasmic (C) or nuclear (N) cell extracts. Quantification of DNA replication activities of the cell extracts was done relative to the CHO K1 NC reaction. Each bar represents the average of four experiments and one standard deviation is indicated. (C) Ku restores replication activity to the xrs-5 nuclear cell extracts. In vitro DNA replication assays were performed with either K1 or xrs-5 nuclear cell extracts in the presence of A3/4-affinity-purified Ku. An autoradiograph of the replication products DNA forms II and III are indicated. Lane 1 and 4, 0 ng affinity-purified Ku (OBA); lane 2 and 5, 160 ng affinity-purified (OBA); lane 3 and 6, 600 ng affinity-purified (OBA). (D) Quantification of DNA replication activities of the cell nuclear extracts, relative to the CHO K1 N reaction using 0 ng of OBA, as described in C. Each bar represents the average of three experiments, and one standard deviation is indicated.

 

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