Figure 3.
Impaired loading of both DNA primase subunits onto early replication origins in the pri2 ( C336A, C417A, C474A ) mutant. Congenic wild-type (PRI1-Myc, PRI2-HA) and mutant (PRI1-Myc, pri2(C336A, C417A, C474A)-HA) cells were synchronized in G1 with α factor and released at 23°C. Cells were collected at the time points indicated. (A) FACS analysis indicates the DNA content of cells throughout the time course. (B) Chromatin-containing extracts were prepared from formaldehyde cross-linked cells collected at the indicated time points. Pri2-HA and Pri1-Myc were immunoprecipitated with anti-HA and anti-Myc monoclonal antibodies, respectively. The recovery efficiency of two early chromosomal replication origins, ARS305 and ARS607, in the immunoprecipitated material relative to the input material was determined by real-time PCR. Background was determined by calculating the amount of target DNA in the mock-IP sample relative to the Input sample (Beads). The results are an average of three independent experiments with standard deviations