Pol2 Is Required for NHEJ Table 1. Repair patterns after a DSB at the URA3::ACT1 intron::HO cut site locus of non-essential DNA polymerase mutants. Imprecise end joininga(%) +CA 2ACA +base 2base Others Chromosomal rearrangementsb(%) insertions deletions mutation Nc 50 20 50 45 20 50 32 37 38 34 28 40 40 45 7.5 70 ndd nd nd nd 73 60 60 55 5 15 nd nd nd nd 0 0 0 0 88 15 nd nd nd nd Nc 40 40 40 40 40 40 nd nd nd nd Strain WT rev3 pol4 pol2-4 pol3-01 pol2-4 pol4 WT (a)d pol2-4 (a) WT (NZ)d pol2-4 (NZ) 28 35 0 69 25 0 66 76 40 74 32 35 92 2.2 40 78 6.3 0 7.9 2.9 50 55 0 76 40 0 66 87 50 85 46 45 98 22 60 100 34 14 47 5.9 2.2 4 0 2 0 0 0 0 2.6 8.8 aAnalysis of survivors grown on YPGal plates. bAnalysis of survivors grown on 5-FOA plates. cTotal events examined. dnd, not determined; a, a factor arrested; NZ, nocodazole arrested. doi:10.1371/journal.pgen.1000060.t001 decrease in the frequency of imprecise end joining events, although the frequency of chromosomal rearrangement events was not significantly affected (Figure 2). Therefore, in agreement with previous findings, Pol4 appears to participate in imprecise end joining repair of NHEJ [15]. A previous study showed that the predominant imprecise NHEJ repair products after an HO-induced DSB at the MATa locus were either addition of two bases (+2 or +CA) or deletion of three bases (23 or 2ACA) [29]. These products are most plausibly caused by a 39-terminal mismatch (HO (+2)) and by a 3 base flap mismatch (HO (23)), respectively (see below for detailed description). Thus, we further analyzed the repair in wild-type and pol4 mutant strains. For wild-type survivors, ,50% had added bases at the junction (including 28% with the +2 repair pattern) and 46% had deleted bases (including 32% with the 23 pattern) (Figure 2C and Table 1). For pol4 cells, however, 98% of survivors had deletion of bases at the junction (including 92% with the 23 pattern), while added bases were not observed in 40 independent colonies examined. This pattern confirms that Pol4 is required for addition of bases during imprecise end joining of NHEJ in vivo [15]. Interestingly, the absolute frequency of repair involving deletion of bases was also decreased more than 3 fold, from 2.5761023 in wild-type cells to 7.2361024 in pol4 cells (Table S1). These results suggest that Pol4 plays a role in imprecise end joining events in vivo where 39flaps are generated. Both Imprecise End Joining and Chromosomal Rearrangement Are Influenced by Essential Polymerases Three essential DNA polymerases (Pol1, Pol2, and Pol3) are required for DNA replication and HR. To investigate whether they also contribute to NHEJ, temperature-sensitive (ts) mutants with defects in polymerase activity were created in the AGY673 background. As shown in Figure 3A, the presence of any of the ts mutations resulted in a .10-fold drop in survival after a DSB, even at the permissive temperature. Despite this, the frequency of survival and pattern of end joining could be compared for each mutant at the permissive and semi-permissive temperatures, and the patterns of end joining could be compared to the WT strain at the equivalent temperature. As a control we analyzed survival of the ts mutants in the absence of a cut site at URA3. We found that strain with no cut sites (STY1553, STY1552 and STY1554) survived equally well at both permissive and semi-permissive temperatures in the absence or presence of HO-endonuclease (Table S2). These results indicate that the change in survival that we observed in the ts mutants after a DSB, was associated with the DSB and its repair, rather than a general effect of the ts mutations, the presence of HO endonuclease, or the growth in galactose versus glucose. Pol1 participates in DNA replication initiation and lagging strand DNA synthesis, as well as HR in yeast [22–24,32]. For Pol1, we found that the frequencies of both imprecise end joining (YPGal/YPD) and chromosomal rearrangements (FOA/ YPD) were reduced more than 50% compared with those cultured at permissive temperature (23uC), a decrease that was significant to a P,0.01 (Figure 3A, 3B, and Table S2). However, there was no significant difference in the pattern of imprecise end joining events or in the percentages of insertions or deletions (Table 2). These results suggest that the effect of Pol1 on NHEJ is indirect. Pol3 is essential for both leading strand and lagging strand DNA synthesis [32]. Interestingly, we found that while the frequency of imprecise end joining did not change, the frequency of chromosomal rearrangements was significantly reduced in the pol3 mutant at the semi-permissive temperature (Figure 3A, 3B, and Table S2). The repair pattern of the pol3 ts mutant showed no obvious difference compared to WT for either end joining or chromosomal rearrangements (Table 3), implying that the major consequence of the pol3 ts mutation at the semi-permissive temperature was a defect in the ability to generate complex chromosomal rearrangements. Pol2 Contributes to Imprecise End Joining through Its 39 to 59 Exonuclease Activity Pol2 interacts with PCNA in S phase and is required for the S- phase checkpoint, for assembly of replication complexes at origins, and for leading strand replication [33,34]. A pol2 polymerase mutant (pol2-18) at the semi-permissive temperature showed no obvious difference in the frequency of imprecise end joining or chromosomal rearrangements compared with the same strain at 23uC (Figure 3A and 3B). Sequence analysis of the imprecise end joining repair pattern, however, revealed that the proportion of 23 events was reduced more than 5 fold at 30uC compared to PLoS Genetics | www.plosgenetics.org 4 April 2008 | Volume 4 | Issue 4 | e1000060