ABSTRACT
BACKGROUND: Heterologous expression of the tumor suppressor BRCA1 in the yeast Saccharomyces cerevisiae is lethal. To identify potential new BRCA1-interacting gene targets, we characterized highly conserved ionizing radiation (IR) sensitive gene deletions that suppress BRCA1-induced lethality in yeast. MATERIALS AND METHODS: Previously, we exposed an isogenic collection of yeast strains individually deleted for 4746 nonessential genes to IR and identified 199 radiation sensitive deletion strains. A subset (n = 130) of these were screened for those that suppressed the G1 arrest and lethality observed following galactose-induced expression from a GAL::BRCA1 plasmid in wild type yeast. RESULTS: We found that deletions of two core components of the highly conserved CCR4-NOT transcription complex (CCR4 or DHH1) rescued BRCA1-induced G1 arrest and lethality in yeast. This was not because of down regulation of the GAL promoter since both deletion strains produce large amounts of BRCA1 that is rapidly degraded. In addition, heterologous expression of BRCA1 results in increased transcription of the DNA damage-inducible reporter construct DIN::LacZ. Reduced viability following IR and nitrogen starvation was observed among strains deleted for CCR4 or DHH1 because of a defect in G1 to S phase checkpoint transition. Lethality following nitrogen starvation and IR was partially rescued in dhh1Delta strains by expressing the human ortholog of DHH1 (DDX6) which has been identified as a breakpoint oncogene.T CONCLUSIONS: hese results suggest that BRCA1 may promote genomic stability in human cells by interacting with the highly conserved ortholog of DHH1 (DDX6) to properly activate G1/S checkpoint arrest following DNA damage.
Subject(s)
DNA Damage/genetics , Gene Expression/genetics , Genes, BRCA1/physiology , Genes, cdc/physiology , Interphase/genetics , RNA Helicases/genetics , RNA-Binding Proteins , Saccharomyces cerevisiae Proteins/genetics , Amino Acid Sequence/genetics , DEAD-box RNA Helicases , Gene Deletion , Genes, Reporter/genetics , Lac Operon/genetics , Nitrogen/metabolism , Promoter Regions, Genetic/genetics , Ribonucleases/genetics , Transcription Factors/geneticsABSTRACT
Human chromosomal DNA contains many repeats which might provide opportunities for DNA repair. We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12). An Alu-URA3-YZ sequence was targeted to several Alu sites within the YAC in strains of the yeast Saccharomyces cerevisiae; the strains contained a galactose-inducible HO endonuclease that cut the YAC at the YZ site. The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retention of the telomeric markers, through recombination between surrounding Alus. For two YACs, the DSBs were not repaired and there was a G2 delay associated with the persistent DSBs. The presence of persistent DSBs resulted in cell death even though the YACs were dispensable. Among the survivors of the persistent DSBs, most had lost the YAC. By a pullback procedure, cell death was observed to begin at least 6 h after induction of a break. For YACs in which the DSB was rapidly repaired, the breaks did not cause cell cycle delay or lead to cell death. These results are consistent with our previous conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C. B. Bennett, A. L. Lewis, K. K. Baldwin, and M. A. Resnick, Proc. Natl. Acad. Sci. USA 90:5613-5617, 1993). However, a break in the YZ-CEN plasmid did not induce lethality in the strain (CBY) background used in the present study. The differences in survival levels appear to be due to the rapid degradation of the plasmid in the CBY strain. We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be long-lived.
