DNA replication error-induced extinction of diploid yeast

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• Genetic defects in DNA repair mechanisms like DNA polymerase accuracy, proofreading, and mismatch repair (MMR) can lead to mutator phenotypes.
• These mutator phenotypes accelerate adaptation in microbes and tumor cells, with certain combinations of mutations driving haploid cell extinction.
• The maximum mutation rate tolerated by diploid cells remains largely undefined.

Purpose of the Study:

• To determine the threshold for replication error-induced extinction (EEX) in diploid Saccharomyces cerevisiae.
• To investigate the synergistic effects of different DNA repair defects on mutation rates and cell viability.
• To explore the role of the S-phase checkpoint kinase Dun1 in suppressing mutator phenotypes and lethality.

Main Methods:

• Creation of double-mutant pol3 alleles with defects in DNA polymerase-δ proofreading and accuracy.
• Analysis of heterozygous and homozygous states of these pol3 alleles in diploid yeast.
• Introduction of antimutator eex mutations and MMR defects.
• Assessment of cell cycle arrest, mutation rates, and growth impairment.
• Inactivation of the Dun1 S-phase checkpoint kinase.

Main Results:

• Heterozygous diploids with specific pol3 double-mutant alleles exhibited strong mutator phenotypes.
• Homozygous pol3-01,L612M and pol3-01,L612G alleles were lethal, causing cell cycle arrest after several divisions.
• Antimutator eex mutations suppressed the lethality of these pol3 alleles.
• MMR defects synergized with certain pol3 alleles, increasing mutation rates and reducing growth.
• Dun1 kinase inactivation suppressed both mutator phenotypes and the lethal pol3-01,L612M phenotype.

Conclusions:

• The lethal error threshold for diploid cells is approximately 10 times higher than for haploid cells, likely linked to homozygous inactivation of essential genes.
• Significant fitness decline occurs at mutation rates substantially lower than the lethal threshold.
• These findings suggest that cancers driven by mutator phenotypes might be treatable with drugs that increase replication errors.