Pol2 Is Required for NHEJ Figure 3. Measurements of NHEJ ability of essential DNA polymerases. The NHEJ assay was performed for pol1, pol3, pol2-18 and pol2-18 pol4 at permissive (23uC) and semi-permissive (30uC) temperatures. (A) Frequencies of imprecise end joining. The survival frequency was evaluated by the ratio of YPGal/YPD. (B) Frequency of chromosomal rearrangement was evaluated by the ratio of FOAR/YPD. Each experiment was collected from at least five independent clones. Dark gray bars indicate 23uC incubation and light gray bars indicate 30uC incubation. *: P,0.05; **: P,0.01. (C) Survivors of pol2-18 or pol2-4 mutants from YPGal plates at 30uC were examined. Bases underlined indicate insertion or mutation. The numbers of inserted (+) and deleted (2) bases were denoted in brackets. N indicates the events of each repair pattern. doi:10.1371/journal.pgen.1000060.g003 23uC, while the proportion of +2 events was nearly doubled (Table 3). These data suggest that Pol2 is particularly involved in generating the 23 product during imprecise end joining repair. A likely step in the creation of the 23 end joining product is deletion of the 3 base 39 flaps from both broken ends. Pol2 might directly participle in this step through its associated exonuclease activity. To examine this possibility, we constructed pol2-4 and pol3-01 mutant strains which are defective in the 39 to 59 exonuclease activity but do not affect the DNA polymerase activity of Pol2 and Pol3, respectively [35,36]. These are both non-lethal mutant strains and both have been shown to display mutator phenotypes [35,37]. Interestingly, pol2-4 but not pol3-01 affects the frequency of imprecise end joining (Figure 2A), and a reduction of the 23 pathway was observed in the pol2-4 strain, but not in the pol3-01 strain (Figure 3C and Table 1). The absolute frequency of base loss during repair was decreased more than 10 fold, from 2.5761023 in wild-type cells to 1.8361024 in pol2-4 cells (Table S1). In particular the absolute frequency of the 23 product was decreased over 98-fold, from 1.7961023 in wild-type cells to 1.8261025 in pol2-4 cells (Table S1). These results support our PLoS Genetics | www.plosgenetics.org 5 April 2008 | Volume 4 | Issue 4 | e1000060