Figure 4.

The Dia2 protein binds replication origins. (A) Chromatin immunoprecipitation was performed on the Dia2-18×myc–tagged strain and an untagged strain. After formaldehyde cross-linking, cells were harvested and lysates prepared. Equal amounts of total protein were subjected to immunoprecipitation with anti-myc antibodies. Chromatin bound during the reaction was isolated and cross-links were reversed before PCR analysis. Primers specific to ARS305 (an early-firing origin), ARS603 (a late-firing origin), and ATP11 (a nonorigin control) were used for PCR. (B) Skp1 and Cdc53 bind origins. Chromatin immunoprecipitation was performed as in A using anti-Skp1 and anti-myc antibodies in a WT strain carrying a Cdc53-myc–tagged vector. (C) Dia2 binds replication origins in a cell cycle–dependent manner. Chromatin immunoprecipitation was performed as in A except that cells were arrested in G1-, S-, and M-phase by treatment with alpha factor, HU, and nocodazole, respectively.