Pol2 Is Required for NHEJ Figure 2. Measurements of NHEJ ability of non-essential DNA polymerases. The efficiency of NHEJ of wild-type and polymerase defective mutants, rev3, pol4 and 39 to 59 exonuclease defective mutants, pol2-4, pol3-01, pol2-4 pol4, dnl4, and pol2-4 dnl4 were measured. (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 four independent clones. *: P,0.05; **: P,0.01. (C) The sequence analysis of the imprecise end joining events from the WT and pol4 mutants. The structures of various imprecise end joining repairs were identified at the DSB site. Sequences of the HO cut site are shown, with the resulting 39 overhanging terminal AACA shown on the cut site. Samples of independent joints were PCR amplified and sequenced. A total of 50 independent survivors on YPGal plates from WT and pol4 mutant 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.g002 URA3 point-mutations) was estimated as the ratio of colonies growing on FOA-containing media compared to colonies growing on YPD, because insertion or deletion of large fragments results in disruption of splicing and consequent FOA resistance. Addition- ally, chromosomal translocations and inversions can also disrupt the integrity of the URA3 gene and cause FOA resistance [31]. It should be noted that the level of analysis carried out in this work (Figure S1) does not definitively distinguish translocations and inversions from insertions. We first compared cell survival after a DSB in strains with or without disruption of nonessential polymerases. The wild-type strain, AGY673, had a frequency of imprecise end joining and chromosomal rearrangement events of 5.5861023 and 1.2761025 per plated cell, respectively (Figure 2A, 2B, and Table S1). Elimination of Rev3 (rev3) did not alter the frequency of imprecise end joining (6.3361023) or chromosomal rearrangement events (1.3361025), suggesting that Rev3 is not required for the NHEJ pathway. In contrast, elimination of Pol4 resulted in an 8.6-fold PLoS Genetics | www.plosgenetics.org 3 April 2008 | Volume 4 | Issue 4 | e1000060