FIGURE 1:

Defect at an early step of DNA replication in the cdc20-ct1 temperature-sensitive mutant. (A) Schematic representation of the Cdc20, the catalytic subunit of Pol ε in S. pombe. Locations of the conserved B-family DNA polymerase domain (polymerase) and cysteine-rich metal binding motifs (metal binding) are shown. The numbering refers to the amino acid residues in the Cdc20 polypeptide. The locations of amino acid alterations in the cdc20-ct1 are shown. (B) Temperature-sensitive growth of the cdc20-ct1 mutant. Tenfold serial dilutions of wild-type and cdc20-ct1 (HM1317) mutant cells were spotted onto EMM plates, and the plates were then incubated at 25°C, 33°C, or 36°C. (C) For synchronous release from M-phase at the restrictive temperature of cdc20-ct1, HM2970 nda3-KM311 (wild-type) and HM1320 cdc20-ct1 nda3-KM311 (cdc20-ct1) cells were arrested at metaphase by incubation at 20°C for 4 h, and then released at 36°C (Time 0). Wild-type cells were released in the absence (wild-type -HU) or presence of HU (15 mM) (wild-type +HU). (D) Aliquots of cells were taken at the indicated time points and analyzed by flow cytometry. Positions of 1C and 2C DNA contents are shown. (E) For synchronous release from HU arrest, wild-type (HM2970) and cdc20-ct1 (HM1320) cells were arrested at metaphase by incubation at 20°C for 4 h and released at 28°C in the presence of HU (13 mM, –3 h). After cultured with HU for 2 h (–1 h), the cells were incubated at 36°C for 1 h and then released into fresh medium without HU (Time 0). (F) Aliquots taken at indicated time points were analyzed by flow cytometry. Positions of 1C and 2C DNA contents are indicated.