Materials and Methods Yeast Strain and Plasmid Constructions General yeast manipulations were performed as described [43]. The S. cerevisiae strains used in this study are listed in Table 3. Yeast strains used in the study were derivatives of AGY628 or AGY673 [44]. The fragments of POL4 (coding sequence 286,969) and REV3 (coding sequence 2652,3863) were PCR- amplified from yeast genomic DNA and cloned into PvuII digested pRS304. pRS304pol4 and pRS304rev3 were linearized by AflII and HpaI, respectively. And these linearized YIPs were transformed into AGY673 using single crossover approach [43] to create pol4 (STY891) and rev3 (STY890) mutant strains. pSD218 [45] (kindly provided by Dr. Daniel E Gottschling) was used as previously described to create the pol1(cdc17-1) mutant in AGY673 (STY969). pST738 was constructed by ligating PCR- amplified pol3(cdc2-2) (coding sequence 2600,1950) from yeast strain UCC5898 (generously provided by Dr. Daniel E Gottschl- ing) [45] to pRS306. BglII linearized pST738 was used to transform AGY628. pol3 of AGY628 was obtained by the two step pop in and pop out method, selecting first for Ura+ and then for Ura2 by FOA and screened for temperature-sensitivity at 37uC for pol3 of AGY628. pol3 of AGY628 was then transformed with the PCR fragment of URA3::ai::HO to create the pol3 ts mutant of AGY673 (STY970). The pol2-18 mutant strain (STY966) was constructed by backcrossing YAH322 [46] (kindly provided by Dr. Akio Sugino) with AGY673 three times, each time selecting for spores with temperature sensitivity. YIpBI and YIpAM26 (kindly provided by Dr. Akio Sugino) were used to create pol2-4 (STY1358) and pol3-01 (STY1359) mutants respectively, as previously described by selecting Ura+ transfor- mants using the single crossover replacement method, and then FOA selection for popouts removed the wild-type sequence [37,43]. Genotype was confirmed by PCR and sequencing. STY1356 (pol2-18 pol4) and STY1360 (pol2-4 pol4) were constructed by disrupting POL4 in STY966 and STY1356 using pRS304pol4, respectively. A ura3 deletion fragment was PCR amplified from an FOA-resistant clone of AGY673. ‘‘No cut’’ site controls were created using the double crossover approach [43] by transforming the ura3 deletion fragment into STY969, STY973 and STY966 and selecting for intergrants on FOA plates to obtain STY1553, STY1552 and STY1554, respectively. The dnl4 strain was constructed by transformation of a PCR product into AGY673 and STY1358 using the genomic DNA from a BY4741 dnl4 strain (Invitrogen) as a template and oligonucleotides flanking the DNL4 gene as primers. Transfor- mants with double crossover at DNL4 were selected for G418 resistance and insertion sites were confirmed by PCR. All primer sequences for PCR are available upon request. Media and Growth Conditions Yeast cells were grown in yeast extract-peptone-dextrose (YPD) or synthetic complete media (SC) with appropriate amino acids missing [47]. Yeast extract-peptone-galactose (YEP-galactose) and yeast extract-peptone-raffinose (YEP-raffinose) contain 2% galac- tose (w/v) and 2% raffinose (w/v), respectively, instead of dextrose (2%). 5- fluoro-orotic acid (5-FOA) plates are SC glucose plates supplemented with 1 mg/ml of 5-FOA [48,49]. The NHEJ Assay The NHEJ assay system was established in the Gabriel lab [28]. This system contains a positive selection for chromosomal changes, including insertions, deletions, translocations and inversions, associated with repair of a defined chromosomal Pol2 Is Required for NHEJ DSB. As shown in Figure 1, a functional URA3 allele was created on chromosome V, which contains a copy of the ACT1 intron as well as the Y–Z junction from the MATa locus. The Y- Z junction includes the recognition sequence and cleavage site for the HO endonuclease, was created on chromosome V. All MAT related sequences had been deleted and this strain contains an integrated galactose-inducible HO endonuclease gene. Induction of the HO Endonuclease, Measurement of DSB Repair Efficiency (Survival Frequency), and 5-FOA Resistance Frequency For non-essential polymerase mutants, multiple independent colonies from each strain were grown at 30uC in YEP-raffinose liquid medium to a final concentration of OD595 ,1. Appropriate dilutions of cells were then plated on YPD or YEP-galactose (YPGal) plates. Colonies were counted after 4 days of growth. Colonies on the YEP-galactose plates were replica plated onto synthetic complete 5-FOA-containing media to measure the frequency of 5-FOA resistance among survivors of HO endonu- clease induction. For essential polymerase mutants, multiple independent colonies from each strain were grown at 23uC in YEP-raffinose liquid medium to a final concentration of OD595 ,1. Appropriate dilutions of cells were then plated on YPD or YPGal plates and incubated at 23uC or 30uC. Colonies were counted after four days of growth. Alternatively, yeast cells were diluted to YPGal liquid medium to OD595 ,0.5, incubated at 23uC or 30uC for 20 hours, and then serial diluted to plate on YPGal and FOA plates. The frequency for imprecise end joining is the ratio of the number of colonies growing on YPGal vs. YPD from per ml of culture. The frequency of chromosomal rearrangements is the number of colonies growing on 5-FOA vs. YPD from per ml of culture. It was notable that colony sizes were smaller and much more heterogeneous on YPGal and FOA plates from all ts mutants, at both the permissive and semi-restrictive temperatures, necessitating an arbitrary cutoff for tiny colonies that were hard to score. All values are expressed as means (6) standard error. Differences between groups were tested using the student’s t- test. For non-essential polymerase mutants, significance tests were compared against frequency of wild type. For essential polymerase mutants, significance tests were compared against frequencies at permissive temperature. Samples of survivors growing on YPGal were analyzed by PCR using primer RAG512 and RAG515 flanking the URA3 gene (Figure 1) [28], and PCR products were sequenced using internal primer RAG513 or RAG633 (Figure 1) [28]. Survivors growing on FOA were further analyzed by Southern blot analysis (Figure S1). For preparing probes, the URA3::ACT1 intron::HO cut site fragment was amplified by PCR using primer RAG512 and RAG515. The 799 (probe A) and 878 (probe B) base pairs XhoI- digested PCR fragments were used as probes for Southern hybridization. In most cases, 40 survivors from four to seven independent cultures were examined for each strain. Based on Southern blot analysis, a rearrangement was termed an insertion if we observed two or more hybridizing bands, a deletion if we observed only a single band, and a mutation if the single band was the same size as the parent strain. Note that potential translocations or inversions were read as insertions. Further, very small insertions could be mis-read as mutations because of minimal change in band size, although we did not see evidence of this after sequencing twenty same size products from the pol3- 01 strain (data not shown). PLoS Genetics | www.plosgenetics.org 8 April 2008 | Volume 4 | Issue 4 | e1000060