23 30 23 30 23 30 23 30 23 30 15 40 35 40 25 30 42 71 0 0 25 25 35 35 40 35 28 4.7 80 74 35 40 50 60 45 40 64 83 0 0 65 50 50 40 55 60 36 17 100 100 0 10 0 0 0 0 0 0 0 0 aAnalysis of survivors grown on YPGal plates. bAnalysis of survivors grown on 5-FOA plates. cTotal events examined. doi:10.1371/journal.pgen.1000060.t002 hypothesis that the 39 to 59 exonuclease activity of Pol2 is involved in the processing step that degrades the 39 flaps to create the 23 end joining product. Further, we confirmed that the events we observed in the pol2-4 strain were in fact due to NHEJ. We compared survival in isogenic dnl4 and pol2-4 dnl4 yeast cells relative to the wild type and pol2-4 strains (Figure 2 and Table S1), and found an ,100 fold drop in the absence of dnl4, a key component of the NHEJ pathway. Of note, we also observed that Table 3. Yeast strains used in this study. WT pol1 pol3 pol2-18 pol2-18 pol4 Strain AGY628 AGY673 YHA322 STY891 STY890 STY969 STY970 STY966a STY1358 STY1359 STY1356b STY1360c STY1544 STY1546 STY1549 STY1550 STY1551 STY1552 STY1553 STY1554 Table 2. Repair patterns after a DSB at the URA3::ACT1 intron::HO cut site locus of essential DNA polymerase mutants. Strain Tm(uC) Imprecise end joininga(%) +CA 2ACA +base 2base Others Chromosomal rearrangementsb(%) insertions deletions mutation Pol2 Is Required for NHEJ 0 0 0 0 0 0 18 5 33 30 Source [44] [44] [46] This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Nc 20 20 40 40 40 40 40 40 40 40 Nc 20 20 20 20 20 20 50 42 50 50 75 50 55 60 80 80 75 88 60 53 25 50 45 40 20 20 7 7 7 17 87.5% of FOA resistant survivors in pol3-01 cells showed the parental size product at the DSB site (Table 1). Sequencing results of the HO cut site junctions in twenty independent survivors revealed that the junctions of each survivor contain different imprecise end joining sequences, suggesting that they are not siblings from a single event (data not shown). Since these changes should not affect splicing of the ACT1 intron, the observed FOA resistance likely resulted from mutations in the URA3 coding Genotype ho,hml::ADE1,mata::hisG, hmr::ADE, ade1, lys5, trp1::hisG, ade3::GAL-HO, ura3-52, LEU2, intronless ACT1 AGY628 URA3::ai::HO cut site MATa pol2-3::LEU2 leu2-3,112 ura3-52 trp1-289 his4 [YCppol2-18] AGY673 pol4::TRP1 AGY673 rev3::TRP1 AGY673 pol1 (cdc17-1) AGY673 pol3 (cdc2-2) AGY673 pol2::LEU2 [YCppol2-18] AGY673 pol2-4 AGY673 pol3-01 AGY673 pol2::LEU2 [YCppol2-18] pol4::TRP1 AGY673 pol2-4 pol4::TRP1 AGY673 dnl4::KanMX4 AGY673 pol2-4 dnl4::KanMX4 AGY673 pol3 (cdc2-2) ura3D AGY673 pol1 (cdc17-1) ura3D AGY673 pol2::LEU2 [YCppol2-18] ura3D AGY673 pol3 (cdc2-2) URA3::ai AGY673 pol1 (cdc17-1) URA3::ai AGY673 pol2::LEU2 [YCppol2-18] URA3::ai aFrom three backcrosses of YHA322 with AGY673. bpol4 of STY966. cpol4 of STY1358. doi:10.1371/journal.pgen.1000060.t003 PLoS Genetics | www.plosgenetics.org 6 April 2008 | Volume 4 | Issue 4 | e1000060