Analysis of DNA replication phenotypes. (A) Plasmid loss rates were measured in orc5-1 hat1, orc5-1, hat1, and wild-type control strains by growing the cells for approximately 10 generations. Strains were JRY2334 (W303 wild-type control), BSY528 (hat1), BSY535 (orc5-1), BSY538 (orc5-1 hat1). These strains were transformed with plasmids pDK243, pDK368-1, pDK368-2, pDK368-7, containing 1, 2, 3 or 8 ARS sequences, respectively. Y-axis shows plasmid loss rates (loss frequency/generation). (B,C) Structural analysis of replicative intermediates by two-dimensional gel electrophoresis. Cultures of hat1 (BSY528) and wild-type (JRY2334) strains were released from G1 arrest for 15 and 30 min (30°C). The same blot was first hybridized with an ARS305 specific probe (B), then stripped and rehybridized with an ARS1 specific probe (C). The 15 min wild-type sample is overloaded as judged by the amount of monomers. (D) Scheme for replicative intermediates observed in two-dimensional gel electrophoresis.