region. These data support the previously described mutator phenotype of pol3-01 [35,36]. Since both Pol2 and Pol4 affect the frequency and pattern of imprecise end joining after an HO-induced DSB, we next asked whether their effects were additive. To this end we tested both pol2-18 pol4 and pol2-4 pol4 double mutant strains with our assay (Figures 2 and 3, and Tables 2 and 3). Strikingly, both pol2-18 pol4 and pol2-4 pol4 double mutant strains showed further reduction of imprecise end joining events compared to those of the single mutations (Figures 2A and 3A). Examining the pattern of repair, we found that the 23 product predominated in the double mutants (Tables 2, 3, S1 and S2). These results suggest that the +2 product pathway is completely Pol4-dependent. The absolute frequency of repair with deletions decreased from 7.2361024 in pol4 cells to 2.5861024 in pol2-4 pol4 cells (Table S1). Therefore, in contrast to Pol4, Pol2 is not the exclusive nuclease for processing the 23 product pathway. Our data also provide evidence for interplay between Pol4 and Pol2 in base deletion and addition during imprecise end joining repair. Although Pol4 has its major effect on base addition, the absolute frequency of base deletion products decreased 3.6 fold, from 2.5761023 in wild-type cells to 7.2361024 in pol4 cells. Conversely, whereas Pol2 is associated with base deletions, the absolute frequency of base addition products decreased .44 fold, from 2.7961023 in wild-type cells to 6.2461025 in pol2-4 cells (Table S1). Lack of Cell Cycle Dependence of Pol2-Mediated 39-End Processing of NHEJ Moore and Haber [29] have shown that specific NHEJ repair products have a cell cycle dependence, with the proportion of 23 products increasing and the proportion of +2 products decreasing in G1. One possible explanation for our observed Pol2-mediated 39- end processing defect is that the cell cycle in the pol2 mutants is altered, possibly even arrested at some stage. In that case, the decreased proportion of 23 products could be related to the cell cycle stage, rather than a direct consequence of Pol2’s role in NHEJ. To test whether Pol2-mediated 39-end processing of NHEJ varies by cell cycle stage, we induced HO cleavage after arresting wild-type or pol2 cells either at G1 or G2/M. As shown in Tables 2 and S1, pol2-4 cells exhibited a reduced frequency of repair via base deletion, regardless of whether the cells were non-synchronized, arrested in G1 or arrested in G2/M. These results indicate that reduction of base deletion during NHEJ in pol2 cells is not cell cycle dependent. Discussion From yeast to humans, NHEJ plays a role in the repair of DSBs. The range of proteins required for the various forms of NHEJ, however, have not yet been defined. DNA polymerases should be important contributors to DNA repair. Gaps often occur which require reconstruction and the nuclease activities associated with DNA polymerases can be utilized for processing repair interme- diates. The essential DNA polymerases Pol1, Pol2 and Pol3 have previously been implicated as being required for HR-type DSB repair [22–24]. Pol4 was the first DNA polymerase found to participate in repairing 39 overhangs during NHEJ in yeast [15]. Interestingly, two Pol4 homologs, Pol m and Pol l, have also been implicated in NHEJ in humans [27,38,39]. The fact that Pol4 directly interacts with DNA ligase IV implies that Pol4 might recruit other NHEJ factors to the DSB sites [11,40,41]. Indeed, we have observed, by chromatin immunoprecipitation, that Pol4 is present at DSB sites (Tseng and Teng, unpublished observation). In this study, we aimed to clarify the in vivo roles of DNA polymerases in NHEJ-type DSB repair. Of the DNA polymerases we examined, only Rev3, showed no involvement in some aspect of Pol2 Is Required for NHEJ NHEJ. This exception is interesting in that Rev3 has been shown, by chromatin immunoprecipitation, to localize to the site of HO- induced DSBs [42]. Pol4 is specifically required for adding bases during imprecise end joining; Pol3 is required for some unknown aspect of generating complex chromosomal rearrangements; both Pol1 and Pol2 play roles in both imprecise end joining and chromosomal rearrangements. Although Pol4has a primary rolein resynthesizing gaps and Pol2contributes to deleting the flaps at imprecise pairing sites at a DSB, we found that Pol4and Pol2can also influence, respectively, base deletion and addition during imprecise end joining repair. These results provide evidence for interplay between Pol4 and Pol2. We speculate that eliminating either one of these polymerases may influence the ability of the other to repair the DSB. This may also account for the observed overall reduction in survival in the presence of any of the ts mutations, even at the permissive temperature (Figure 3A). Even subtle changes in these essential proteins could tip the balance away from successful repair. Our study reflects the diversity, collaboration, and redundancy of multiple DNA polymerases, both essential and non-essential, in eukaryotic repair processes. DSBs generate different DNA end structures, which need to be recognized and repaired by complexes that likely include more than one DNA polymerase. Our data demonstrates that Pol2, and more specifically, the 39 to 59 exonuclease activity of Pol2, plays a significant role in generating imprecise NHEJ joints that require removal of 39 terminal flaps (Figure 4). However, unlike filling in gaps resulting from 39 terminal mismatches, which seems to be an exclusive function of Pol4, flap removal can be carried out by nucleases other than Pol2, although at reduced efficiency. The frequencies of imprecise end joining in pol2-18 pol4 and pol2-4 pol4 double mutant strains were significantly decreased compared to those of wild-type or either single mutant, but in both double mutant strains DSB repair was predominantly through the 23 pathway of imprecise end joining. Since the pol4 mutant used in this study contains a complete truncation of the POL4 open reading frame, our data imply that other unidentified 39 to 59 exonucleases might be utilized in this NHEJ process. Further studies with additional mutant genes should help to delineate the roles and compartmen- talization of particular repair factors in different aspects of NHEJ. Figure 4. Proposed pathways for imprecise end joining of the HO endonuclease induced DSB at MATa. The 4-bp 39 overhanging sequence resulting from HO endonuclease cleavage is indicated. The two ends must be brought together into synapsis. Precise religation would lead to re-cutting. Possible end processing and subsequent alignment of complementary base pairs that would lead to insertions or deletions are shown. When 39 overhangs mispair, Pol4 is required to fill in the gap [15,16]. When 39 flaps are generated by mis-alignment, the 39 to 59 exonuclease activity of Pol2 is the predominate exonuclease to remove the flaps. doi:10.1371/journal.pgen.1000060.g004 PLoS Genetics | www.plosgenetics.org 7 April 2008 | Volume 4 | Issue 4 | e1000060