Fig. 1
Pol ε catalytic domains are critical for yeast growth. a Schematic representation of DNA Polymerase ε (Pol ε). The S. cerevisiae holoenzyme consists of the catalytic subunit (Pol2p) and three auxiliary subunits: Dpb2p, Dpb3p, and Dpb4p52,53,54,55. Cryo-electron microscopy has shown that Pol2p has two lobes tethered by a flexible linker43. Active polymerase and exonuclease domains are in the N-terminal lobe. The pol2-16 mutant has an in-frame deletion of the fragment of catalytically active lobe (amino acids 176–1134). b, c Tetrad analysis of pol2-16/POL2 and pol2-4/POL2 heterozygous diploids in two yeast backgrounds, ∆7, and W303, at 23 °C b and 30 °C c. 1–12 are dissected tetrads, A–D, and a–d are haploid spore colonies. Images were taken after 3 and 12 days. Genotypes were confirmed via PCR (pol2-16, red circles) or sequencing (pol2-4, blue). Wild-type colonies are circled in green. The lack of both Pol ε catalytic domains (pol2-16) causes severe growth defects. Exonuclease inactivation alone (pol2-4) does not. d Microscopic images of pol2-16 colonies taken 3 days after tetrad dissections. e Doubling times of pol2-16 and pol2-4 mutants compared to wild-type yeast. Doubling times were estimated from optical density at 600 nm of cultures grown at 23 °C. Error bars represent standard deviations (n = 4–6 yeast cultures, two or three from two independent isolates). Unpaired two-tailed t tests with Welch’s correction yielded p values (P). The doubling time of the pol2-16 mutant is about threefold longer than of the wild-type and pol2-4 yeast in both ∆7 and W303 backgrounds. The difference in the doubling times between the wild-type ∆7 and W303 backgrounds may be due to one or more of over 10,000 SNPs detected by the whole-genome sequencing. f Western blot detection of Pol2p level in whole-cell extracts. Presented are bands for three independent isolates of strains bearing POL2 or pol2-16 in fusion with TAP-tag. Immunoblotting was performed using an antibody to TAP-tag or PSTAIR (loading control). g Relative band intensity. Error bars represent standard deviations (n = 6–7 independent yeast isolates). Unpaired two-tailed t tests with Welch’s correction yielded p values (P)
