Characterization of mutations that are synthetic lethal with pol3-13 , a mutated allele of DNA polymerase delta in Saccharomyces cerevisiae

Abstract

The pol3-13 mutation is located in the C-terminal end of POL3, the gene encoding the catalytic subunit of polymerase delta, and confers thermosensitivity onto the Saccharomyces cerevisiae mutant strain. To get insight about DNA replication control, we performed a genetic screen to identify genes that are synthetic lethal with pol3-13. Mutations in genes encoding the two other subunits of DNA polymerase delta (HYS2, POL32) were identified. Mutations in two recombination genes (RAD50, RAD51) were also identified, confirming that homologous recombination is necessary for pol3-13 mutant strain survival. Other mutations were identified in genes involved in repair and genome stability (MET18/ MMS19), in the control of origin-firing and/or transcription (ABF1, SRB7), in the S/G2 checkpoint (RAD53), in the Ras-cAMP signal transduction pathway (MKS1), in nuclear pore metabolism (SEH1), in protein degradation (DOC1) and in folding (YDJ1). Finally, mutations in three genes of unknown function were isolated (NBP35, DRE2, TAH18). Synthetic lethality between pol3-13 and each of the three mutants pol32, mms19 and doc1 could be suppressed by a rad18 deletion, suggesting an important role of ubiquitination in DNA replication control. We propose that the pol3-13 mutant generates replicative problems that need both homologous recombination and an intact checkpoint machinery to be overcome.

Replication Stress Leads to Genome Instabilities inArabidopsisDNA Polymerase Δ Mutants

“…To explain the increased HR phenotype of pold1 plants, however, we favor a hypothesis according to which the DNA breaks are directly created by uncoordinated and faulty DNA synthesis due to the lack of replication factors. This mode of action was previously suggested to be the cause of gross chromosomal instabilities observed in budding yeast (Saccharomyces cerevisiae) (Chanet and Heude, 2003;Galli et al, 2003) and mouse (Mus musculus) cells (Venkatesan et al, 2007), carrying mutations in the catalytic subunit of Pold.…”

Conserved electron donor complex Dre2–Tah18 is required for ribonucleotide reductase metallocofactor assembly and DNA synthesis

“…Fe-S cluster-binding domains have been found in an increasing number of nuclear proteins involved in DNA replication and repair including DNA primase, DNA helicases, and DNA polymerases (54). The importance of Dre2-Tah18 in DNA replication also was supported by the synthetic lethality between a ts mutant of POL3 encoding the Fe-S cluster containing DNA polymerase δ and dre2 and tah18 mutant alleles (55). Our findings show that the Dre2-Tah18 complex is required for assembly of the di-iron enzyme RNR cluster and for cellular supplies of dNTP.…”

Dre2, a Conserved Eukaryotic Fe/S Cluster Protein, Functions in Cytosolic Fe/S Protein Biogenesis

“…Synthetic lethal interaction with pol3-13, a mutated allele of DNA polymerase delta, was described previously (6), and a recent publication demonstrated an effect of a temperature-sensitive allele on chromosome transmission fidelity, although a mechanism of this effect has not been described (5). In order to confirm the essentiality of DRE2, a shuffle strain was constructed, and counterselection against the covering plasmid was shown to FIG.…”