Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast.

Abstract

To investigate the mechanisms that ensure the dependency relationships between cell cycle events and to investigate the checkpoints that prevent progression through the cell cycle after DNA damage, we have isolated mutants defective in the checkpoint and feedback control pathways. We report the isolation and characterization of 11 new loci that define distinct classes of mutants defective in one or more of the checkpoint and feedback control pathways. Two mutants, rad26.T12 and rad27.T15, were selected for molecular analysis. The null allele of the rad26 gene (rad26.d) shares the phenotype reported for the "checkpoint rad" mutants rad1, rad3, rad9, rad17, and hus1, which are defective in the radiation checkpoint and in the feedback controls that ensure the order of cell cycle events. The null allele of the rad27 gene (rad27.d) defines a new class of Schizosaccharomyces pombe mutant. The rad27 complementing gene codes for a putative protein kinase that is required for cell cycle arrest after DNA damage but not for the feedback control that links mitosis to the completion of prior DNA synthesis (the same gene has recently been described by Walworth et al. (1993) as chk1). These properties are similar to those of the rad9 gene of Saccharomyces cerevisiae. A comparative analysis of the radiation responses in rad26.d, rad26.T12, and rad27.d cells has revealed the existence of two separable responses to DNA damage controlled by the "checkpoint rad" genes. The first, G2 arrest, is defective in rad27.d and rad26.d but is unaffected in rad26.T12 cells. The second response is not associated with G2 arrest after DNA damage and is defective in rad26.d and rad26.T12 but not rad27.d cells. A study of the radiation sensitivity of these mutants through the cell cycle suggests that this second response is associated with S phase and that the checkpoint rad mutants, in addition to an inability to arrest mitosis after radiation, are defective in an S phase radiation checkpoint.

Chk1 complements the G2/M checkpoint defect and radiosensitivity of ataxia-telangiectasia cells

“…Overexpression of chk1 in wild type S. pombe led to cellular elongation and a failure to enter mitosis (Al-Khodairy et al, 1994;Ford et al, 1994). This was the same phenotype as observed with a number of radmutants suggesting that these function upstream of chk1 in checkpoint arrest (Walworth and Bernards, 1996).…”

“…This is unlikely to be the case since equal amounts of hchk1 protein were detected in AT1ABR and C3ABR but there is a defect in activation of chk1 in AT1ABR cells (Khanna et al, unpublished). Previous data demonstrate that a gainof-function caused by overexpression of chk1 alone in S. pombe acting downstream of the ATM homologue rad3, leads to G2 arrest (Al-Khodairy et al, 1994;Ford et al, 1994;Walworth et al, 1993). In addition moderate overexpression of chk1 in S. pombe rad mutants, at levels that do not e ect mitotic entry, restored partially their level of survival after DNA damage (Walworth et al, 1993).…”

Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint

“…The double mutants between mis3-224 (Takahashi et al e1994) and various mutations noted below were constructed; cdc2-33, cdc6-121, cdc13-117, cdc25-22, cdc10-129 (Nurse et al 1976), cdc2-3w (Enoch et al 1992), Dcyc17/cig2 (Obara-Ishihara & Okayama 1994), Dmik1 (Lundgren et al 1991), wee1-50 (Russell & Nurse 1987), cdc16-116 (Fankhauser et al 1993), cdc17-K42, cdc18-K46 (Nasmyth & Nurse 1981), cdc22-C1 (Fernandez Sarabia et al 1993), swi7-H4 (Singh & Klar 1993), mis5-268, mis11-453 (Takahashi et al 1994, Drad3, Drad9, Drad17, Drad24, Drad25, Dchk1 (Al-Khodairy et al 1994), cut5-T401 (Saka et al 1994), Dcds1 (Murakami & Okayama 1995), Ddis1 (Nabeshima et al 1995), dis3-54 (Kinoshita et al 1991), dis2-11, Ddis2 (Ohkura et al 1989), cut1-21, cut2-364 (Funabiki et al 1996, cut9 (Yamada et al 1997), Dppa1, Dppa2 (Kinoshita et al 1993),Dpck1 (Toda et al 1993), Dtop1, top2-191 (Uemura & Yanagida 1984), and Ddsk1 mutants (Takeuchi & Yanagida 1993). The Mis3-HA integrant was obtained by integrating the HA-tagged mis3 gene at the carboxyl-terminus to replace the genomic mis3 locus.…”

Mitotic DNA damage and replication checkpoints in yeast

“…The checkpoint rad genes include rad1 sp , rad3 sp , rad9 sp , rad17 sp , rad26 sp , and hus1 sp . These are required for all the known S. pombe DNA checkpoints [3,13,14]. At the very least, the checkpoint rad gene products serve as signal transducers between the primary checkpoint signal and the cell cycle machinery.…”