Rad Gene Research Articles

The chk1 pathway is required to prevent mitosis following cell-cycle arrest at ‘start’

The G2-M-phase transition is controlled by cell-cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. Genetic analyses in yeast have defined two related, but distinct, pathways which prevent mitosis--one which acts when S phase is inhibited, and one which acts when the DNA is damaged. In the fission yeast Schizosaccharomyces pombe, many of the gene products involved have been identified. Six 'radiation checkpoint' (rad) gene products are required for both the S-M and DNA-damage checkpoints, whereas Chk1, a putative protein kinase, is required only for the DNA-damage checkpoint and not for the S-M checkpoint following the inhibition of DNA synthesis. We have genetically defined a third mitotic control checkpoint pathway in fission yeast which prevents mitosis when passage through 'start' (the commitment point in G1) is compromized. In cycling cells arrested at start, mitosis is prevented by a Chk1-dependent pathway. In the absence of Chk1, G1 cells attempt an abortive mitosis with a 1C DNA content without entering S phase. Similar results are seen in the absence of Rad17, a typical example of a rad gene product. Genetic dissection of checkpoints in logarithmically growing fission yeast has identified a pathway that couples mitosis to correct passage through start. This pathway is related to the DNA-structure check-points which ensure that mitosis is dependent on the completion of replication and the integrity of the DNA. We propose that all three mitotic control checkpoints monitor distinct DNA or protein structures at different stages in the cell cycle.

Doing the right thing: feedback control and p53

Recent evidence suggests that exposure of cells to DNA-damaging agents causes a rise in the levels of the p53 tumor suppressor protein and arrest of progression through the cell cycle. p53 may therefore resemble a member of the RAD gene class identified in yeast, RAD9, which allows cells to repair DNA before continuation of the cell cycle. The evidence that p53 is a sequence-specific, DNA-binding protein that can regulate transcription suggests several ways in which p53 might effect this growth cessation.

The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae.

The survival of plasmid YRp12 treated in vitro with ultraviolet- or gamma-radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae. Yields of pyrimidine dimers or single and double strand breaks in plasmid DNA were assayed by physical methods. The biological effects of these damages were assayed by transformation of wild-type cells and rad mutants from each of the major groups of radiosensitive mutants. After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival. After gamma-irradiation no such correspondence was found. Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect. Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability. More generally these data also show that transformation events using the LiCl protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of DNA concentrations.

EFFECT OF GENES CONTROLLING RADIATION SENSITIVITY ON CHEMICALLY INDUCED MUTATIONS IN SACCHAROMYCES CEREVISIAE

The effect of 16 different genes (rad) conferring radiation sensitivity on chemically induced reversion in the yeast Saccharomyces cerevisiae was determined. The site of reversion used was a well-defined chain initiation mutant mapping in the structural gene coding for iso-1-cytochrome c. High doses of EMS and HNO(2) resulted in decreased reversion of cyc1-131 in rad6, rad9 and rad15 strains compared to the normal RAD+ strains. In addition, rad52 greatly decreased EMS reversion of cyc1-131 but had not effect on HNO( 2)-induced reversion; rad18, on the other hand, increased HNO( 2)-induced reversion but did not alter EMS-induced reversion. When NQO was used as the mutagen, every rad gene tested, except for rad14 , had an effect on reversion; rad6, rad9, rad15, rad17, rad18, rad22, rev1, rev2 and rev3 lowered NQO reversion while rad1, rad2, rad3, rad4, rad10, rad12 and rad16 increased it compared to the RAD+ strain. The effect of rad genes on chemical mutagenesis is discussed in terms of their effect on UV mutagenesis. It is concluded that although the nature of the repair pathways may differ for UV- and chemically-induced mutations in yeast, a functional repair system is required for the induction of mutation by the chemical agents NQO, EMS and HNO(2).

The effect of genes controlling radiation sensitivity on chemical mutagenesis in yeast.

A special set of mutants containing identified altered codons in the iso-1-cytochrome c gene of yeast were recently used as tester strains for determining mutagenic specificities (Prakash and Sherman, 1973). One of the mutants, cyc1–131, contains a GUG codon in place of the normal chain-initiation codon AUG and requires a G•C-to-A•T transition to yield the normal protein (Stewart et al., 1971). This mutant was reverted preferentially and with a high frequency by ethyl methanesulfonate (EMS), diethyl sulfate (DES), nitrous acid (HNO2), N-methyl-N ′-nitro-N-nitrosoguanidine (NTG), nitrosoimidazolidone (NIL), [3H] uridine ([3H] U), nitroquinoline oxide (NQO), and β-propioloactone (β-PL), and it was therefore concluded that these agents selectively induced G•C-to-A•T transitions (Prakash and Sherman, 1973; Prakash et al., 1974). In addition, ultraviolet light (UV), X-rays, nitrogen mustard (HN2), methyl methanesulfonate (MMS), and dimethyl sulfate (DMS) were found to revert all the tester strains with the same efficiency or without any dependence on simple types of base-pair changes, and it was concluded that these mutagens were nonspecific in the types of base-pair changes produced. Since the cyc1–131 tester reverted well with all 11 chemical mutagens and with ionizing and UV radiation, it was used in studies designed to determine the genetic control of mutation induction using a wide variety of mutagens (Prakash, unpublished results). This tester was coupled to over 20 different rad genes (the symbol for loci conferring radiation sensitivity in yeast), and diploids were constructed which were homozygous for cyc1–131 as well as a particular rad gene.