Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28 gene

DNA polymerase ε (pol ε) participates in the leading DNA strand synthesis in eukaryotes. The catalytic subunit of this enzyme, Pol2, is a fusion of two ancestral B-family DNA polymerases. Paradoxically, the catalytically active N-terminal pol is dispensable, and an inactive C-terminal pol is essential for yeast cell viability. Despite extensive studies of strains without the active N-terminal part (mutation ), it is still unclear how they survive and what the mechanism is of rapid recovery of initially miserably growing cells. Slow progress is attributed to the difficultly of obtaining strains with the defect. We designed a robust method for the construction of mutants with only the C-terminal part of Pol2 using allele optimized for high protein production. Colonies bearing appear three times sooner than colonies of but exhibit similar growth defects: sensitivity to hydroxyurea, chromosomal instability, and an elevated level of spontaneous mutagenesis. UV-induced mutagenesis is partially affected, it is lower only at high doses in some reporters. The analysis of the genomes of isolates revealed the prevalence of nonsynonymous mutations suggesting that the growth recovery was a result of evolution by positive selection for better growth fueled by variants produced by the elevated mutation rate. Mutations in the gene, the primary regulator of the cell cycle, were repeatedly found in independent clones. Genetic analysis established that alleles single-handedly improve the growth of strains and suppress HU-sensitivity. The affected amino acids are located on the Cdc28 molecule’s surfaces that mediate contacts with cyclins and kinase subunits. Our work establishes the significance of the gene for the resilience of replication and predicts that changes in mammalian homologs, cyclin-dependent kinases may play a role in remastering replication to compensate for the defects in the leading strand synthesis by the dedicated polymerase.