Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth.
Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alpha. DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both. The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins. Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity. Only damage-sensitive alleles were lethal in combination with a ctf4 deletion. Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4. Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol alpha and Ctf4, in a role that is optimal with, but does not require, full helicase activity.
Formosa
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Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA. formosa@medschool.med.utah.edu
Nittis
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D000483:Alleles; D001483:Base Sequence; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D009154:Mutation; D010641:Phenotype; D011506:Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016615:Telomere / Q000235:genetics
C078338:CTF4 protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D004256:DNA Polymerase I; D004265:DNA Helicases