FIGURE 1.

Time course of association of replication proteins with two human origins of DNA replication. A, association with replication origins at the LB2 gene (left panels) and at the UPR of the MCM4 gene (right panels) with nucleoprotein complexes isolated by immunoprecipitation of S phase Orc2, Mcm3, and Pol ϵ. HeLa cells were synchronized by double thymidine block to early S phase (0 h) and released to proceed in S phase (2 and 4 h). Nucleoproteins derived from CsCl centrifugation were sonicated and digested with micrococcal nuclease as described under “Experimental Procedures.” Soluble nucleoproproteins, 1 mg/500 μl, were taken for immunoprecipitation with the cognate antibodies, the protein was digested with proteinase K and DNA isolated. From 5 to 10 ng of DNA were obtained from the immunoprecipitate, 1/20 was used for quantitative PCR (precipitate). DNA from soluble nucleoproteins representing genomic DNA was isolated and treated in the similar manner, except that the immunoprecipitation step was omitted, and analyzed by quantitative PCR (input). The values are mean values from two distinct experiments. The regions analyzed by qPCR are indicated in the panels and their locations are shown below the panels, for LB2 origin and UPR origin, see the left and right panels, respectively. The LB2 gene is on the left site of the replication origin as indicated by an arrow. The MCM4 gene is on the left and PRKDC gene on right site of the replication origin as presented by arrows. The MCM4 gene encodes the minichromosome maintenance protein 4 and the PRKDC gene the catalytic subunit of the DNA-dependent protein kinase. The origin regions are shown as gray boxes below the axis. Black bars above the axis represent regions that were amplified by qPCR. B, cell cycle progression of G₁/S-arrested cells released to progress into S phase as verified by FACS of propidium iodine-treated cells.