Fission Yeast Pot1 and RecQ Helicase Are Required for Efficient Chromosome Segregation

Abstract

Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1؉ causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1⌬ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1⌬ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1⌬ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.

A Double Mutant between Fission Yeast Telomerase and RecQ Helicase Is Sensitive to Thiabendazole, an Anti-Microtubule Drug

“…10) In contrast, about 90% of the trt1Á rqh1-hd double mutant had RPA foci on the chromosome bridge during the M phase ( Fig. 3B and C), suggesting that recombination intermediates are present at the chromosome ends in the M phase.…”

“…Accumulation of recombination intermediates at chromosome ends is perhaps the reason for this. 10) Similarly, the chromosome end fragments of the trt1Á rqh1Á double mutants from day 16 of culture did not enter the pulsed-field electrophoresis gel (Fig. 1E, lane 6), suggesting that recombination intermediates accumulate at the chromosome ends in the trt1Á rqh1Á double mutants.…”

“…10) We asked whether the trt1Á rqh1Á double mutant displays similar phenotypes. The trt1Á rqh1Á double mutant from day 16 of culture was used for spot assay (Fig.…”

“…To compare the functional differences between Trt1 and Pot1 directly on a rqh1-hd mutant background, we created a trt1Á rqh1-hd double mutant by same method we used to create the pot1Á rqh1-hd double mutant. 10) First we created the trt1Á rqh1-hd double mutant, in which trt1…”

“…These RPA foci are perhaps recombination intermediates at the chromosome ends, which might be the reason for the TBZ sensitivity. 10) To determine whether the trt1Á rqh1-hd double mutant has RPA foci 15) B, The telomere lengths of wild-type (WT), trt1Á, and trt1Á rqh1Á cells were analyzed by Southern hybridization at 30 C during growth in liquid culture at the indicated days after tetrad dissection. Genomic DNA was digested with NsiI, separated by 0.8% agarose gel electrophoresis, and hybridized to a 1-kbp DNA fragment containing telomere plus TAS1 sequences.…”