Abstract
The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with and is functionally related to the structural maintenance of chromosomes 5 and 6 (SMC5/6) protein complex. In this study, we investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU. Significantly, Rad60 function is not required during HU arrest but is required on release. However, the mutant cells underwent aberrant mitosis accompanied by irregular segregation of chromosomes, and DNA replication was not completed, as revealed by pulsed-field gel electrophoresis. The deletion of rhp51 suppressed the aberrant mitosis of rad60-1 cells and caused mitotic arrest. These results suggest that Rhp51 and Rad60 are required for the restoration of a stalled or collapsed replication fork after release from the arrest of DNA replication by HU. The rad60-1 mutant was proficient in Rhp51 focus formation after release from the HU-induced arrest of DNA replication or DNA-damaging treatment. Furthermore, the lethality of a rad60-1 rqh1Delta double mutant was suppressed by the deletion of rhp51 or rhp57. These results suggest that Rad60 is required for recombination repair at a step downstream of Rhp51. We propose that Rhp51-dependent DNA structures that cannot activate the mitotic checkpoints accumulate in rad60-1 cells.
Highlights
The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks
We investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU
We analyzed the effects of rad60-1, rhp51⌬, and rqh1⌬ mutations on the phenotypes of cells after their release from the arrest of DNA replication caused by HU
Summary
S. pombe cells were grown in yeast extractsupplemented (YES) medium or Edinburgh minimal medium, and standard genetic and molecular procedures were employed as described previously [34]. To examine sensitivity to HU on plates, serial dilutions of cells were spotted on YES medium plates containing various concentrations of HU and incubated at 26°C for 3 days. To examine sensitivity to HU in liquid medium, cells were grown in YES medium to a density of ϳ1 ϫ 107/ml, and HU was added to a concentration of 15 mM. Pulsed-field gel electrophoresis was carried out with 0.5% chromosomal-grade agarose (Bio-Rad) in 1ϫ Tris-acetate-EDTA buffer by using a CHEF Mapper apparatus (Bio-Rad). Cells were fixed with 3.7% formaldehyde and processed as described previously [9]. Distributions of DNA contents of the cells were analyzed by staining cells with propidium iodide and processing them for fluorescenceactivated cell sorter (FACS) analysis as described in reference 41
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