Regulation of Telomere Length by Checkpoint Genes in Schizosaccharomyces pombe

We have studied telomere length in Schizosaccharomyces pombe strains carrying mutations affecting cell cycle checkpoints, DNA repair, and regulation of the Cdc2 protein kinase. Telomere shortening was found in rad1, rad3, rad17, and rad26 mutants. Telomere lengths in previously characterized rad1 mutants paralleled the replication checkpoint proficiency of those mutants. In contrast, rad9, chk1, hus1, and cds1 mutants had intact telomeres. No difference in telomere length was seen in mutants affected in the regulation of Cdc2, whereas some of the DNA repair mutants examined had slightly longer telomeres than did the wild type. Overexpression of the rad1+ gene caused telomeres to elongate slightly. The kinetics of telomere shortening was monitored by following telomere length after disruption of the rad1+ gene; the rate was ∼1 nucleotide per generation. Wild-type telomere length could be restored by reintroduction of the wild-type rad1+ gene. Expression of the Saccharomyces cerevisiae RCK1 protein kinase gene, which suppresses the radiation and hydroxyurea sensitivity of Sz. pombe checkpoint mutants, was able to attenuate telomere shortening in rad1 mutant cells and to increase telomere length in a wild-type background. The functional effects of telomere shortening in rad1 mutants were assayed by measuring loss of a linear and a circular minichromosome. A minor increase in loss rate was seen with the linear minichromosome, and an even smaller difference compared with wild-type was detected with the circular plasmid.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.