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, 1/20^th 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, 1/20^th 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, 1/20^th of DNA recovered from immunoprecipitation with anti-Ku70 anti-Ku86, or anti-clone162 from CHO K1 cross-linked cells.

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