Figure 4

Orc2p and topo II reside at the UPR origin. (A) The UPR origin is located between the divergently transcribed human genes PRKDC and MCM4. Squares above the distance marker indicate the DNA regions analyzed by PCR. The data and the PCR primer are described in [19]. (B) The procedures for chromatin immunoprecipitation (ChIP) and quantitative PCR were decribed in detail [19]. Here we performed ChIP with Orc2p-specific (circles) and, in parallel, with topo II- specific (squares) antibodies. Quantitative PCR was performed with total deproteinized chromatin before precipitation (input) and the immunoprecipitate using the primers indicated in (A). (C) HeLa cells were treated for 90 min with the topo II-specific drug etoposide (from Sigma) in the concentrations indicated. The DNA was extracted and prepared for PCR. The data are expressed relative to the amplification of the exon-9 (Ex9) sequence which carries no topo II (see: panel B). Negative controls are the ratios of amplifiable DNA from exon 2 (Ex2; open square) in the MCM4 gene (see: panel B) and a sequence from an origin-distal part of human gene HBB [26](open circle). Positive control is the lamin B2 origin (LB; filled square) amplified using the primer pair described in [19]. UPR (filled circle) is the origin sequence in the MCM4 gene (see above: A and B).