1. Volume 3, Issue 5, Pages 679-685 (May 1999)
DNA Polymerase ε Catalytic Domains Are Dispensable for DNA Replication, DNA Repair, and Cell Viability 
Tapio Kesti, Karin Flick, Sirkka Keränen, Juhani E Syväoja, Curt Wittenberg 
Molecular Cell 
Volume 3, Issue 5, Pages (May 1999)
DOI: /S (00)

2. Figure 1
The Pol2 Carboxyl Terminus Lacking Catalytic Domains Is Sufficient to Complement Temperature-Sensitive Mutants of POL2
(A) Structure of the Pol2 protein, point mutants, and deletion mutants. The numbering refers to the amino acid residues in Pol2 polypeptide. The positions of conserved 3′→5′ exonuclease and DNA polymerase motifs are shown. The most conserved polymerase motif I is indicated by I. pol2-3 (referred to here as pol2Δ), pol2-18, and pol2-11 mutations have been described previously (Morrison et al. 1990; Araki et al. 1992; Budd and Campbell 1993; Navas et al. 1995). pol2-16 lacks the 3′→5′ exonuclease and DNA polymerase motifs, while pol2ΔC is devoid of the entire carboxy-terminal portion of Pol2. The relevant restriction sites for the Southern analysis presented in Figure 3 are presented at the bottom of the diagram.
(B) The carboxyl terminus of Pol2 (pol2-16) is sufficient to complement temperature-sensitive mutants of POL2. pol2-16 can complement both pol2-11 (C-terminal) and pol2-18 (N-terminal) temperature-sensitive mutants. Yeast cells were transformed with the indicated plasmids, streaked onto SC-uracil plates (Glc), and incubated for 3–4 days at 25°C or 36°C.
(C) GAL-pol2-16, but not GAL-pol2ΔC, can complement both pol2 ts mutants. Yeast cells were streaked onto SC-uracil plates containing galactose (Gal) to induce the GAL promoter and incubated for 3–4 days at 25°C or 36°C. The pol2-18 allele is present in a centromeric plasmid over a pol2 disruption since pol2-18 is insufficient for viability when present in the chromosome in a single copy (our observations; H. Araki and A. Sugino, personal communication).
Molecular Cell 1999 3, DOI: ( /S (00) )

3. Figure 3
Cells Lacking Pol2 Catalytic Domains Are Proficient in Excision Repair and Double-Strand Break Repair
(A) Plating efficiency of pol2 mutants following exposure to UV irradiation. Dilutions of cells were spread on YEPD plates and irradiated with UV light at 254 nm followed by incubation for 3–5 days and counting of colonies. The relevant genotypes of the strains are indicated.
(B) Sensitivity of pol2-16 mutants to HO-induced double-strand DNA breaks. The relative plating efficiency of cells containing the YCp50-GAL-HO (URA3) plasmid (open bars) was determined by plating on SC-uracil containing glucose or galactose. Cells containing a YCp GAL (URA3) plasmid (solid bars) were used as a control. rad52 strains, known to be defective in the double-strand break repair, were included for comparison.
Molecular Cell 1999 3, DOI: ( /S (00) )

4. Figure 2
The Carboxyl Terminus of Pol2 (pol2-16) Is Sufficient for DNA Replication and Cell Proliferation
(A) pol2-16 can complement a complete disruption of POL2. Cells containing a pol2 disruption (pol2-3) were transformed with either a plasmid with wild-type POL2, or pol2-16 expressed from the plasmid indicated. pol2-16 and pol2-11 were expressed from the endogenous chromosomal locus. Yeast cells were streaked onto YEPD (Glc) or YPGal (Gal) plates and incubated at 25°C or 36°C.
(B) Southern analysis was performed using SalI and SphI-digested genomic DNA isolated from the indicated strain probed with a 4.2 kb PstI fragment of POL2. The expected fragments are indicated (see Figure 1). Segregants A–D (lanes 4–7) represent the four viable products dissected from a single tetrad obtained by sporulation of a POL2/pol2Δ trp1/trp1::TRP1::GAL1::pol2-16 diploid (TAY120, lane 1). The Leu and Trp phenotypes of each spore and the parent are indicated below each lane and are consistent with the genotype demonstrated by Southern analysis.
(C) Reduced rate of chromosomal DNA replication and delay in cell cycle progression in pol2-16 mutants. rho0 derivatives of POL2 and pol2-16 strains were synchronized with mating pheromone α factor and released into fresh medium lacking mating pheromone. Samples were taken at the times indicated, and their DNA content was determined by flow cytometry.
Molecular Cell 1999 3, DOI: ( /S (00) )

5. Figure 4
Cells Lacking Pol2 Catalytic Domains Are Viable in the Absence of the MEC1-Dependent Replication and Damage Checkpoints
Equal numbers of wild-type, mec1, pol2-16, and pol2-16 mec1 cells from fresh cultures were streaked onto YPGAL plates with or without 20 mM HU. The plates were incubated at 30°C for 3 days. All strains are cln1Δ cln2Δ, which is required for viability of the mec1-1 mutant in the 15Dau background (Vallen and Cross 1999).
Molecular Cell 1999 3, DOI: ( /S (00) )