Author Summary Chromosomal DSBs caused by replication fork disruption, environmental factors, or endogenous nucleases are common yet potentially dangerous DNA lesions in all organisms. If they are repaired by homologous recombi- nation (HR), the integrity of the genome is usually maintained. However, if the broken ends undergo NHEJ, sequences at the junction may be added, deleted, or substituted, and large segments of chromosomes can be rearranged. Partially overlapping sets of proteins are required for repair by either the HR or NHEJ pathway. Furthermore, different proteins may be used to process broken DNA ends, depending on the particular terminal structures. Since DNA synthesis occurs during HR in yeast and all three essential replicative polymerases are utilized, we asked how different polymerases might be involved in DSB repair by NHEJ. We find that Pol2, and particularly the enzyme’s 39 to 59 nuclease activity, contributes to the removal of 39 single strand flaps that can form during the initial joining of broken ends. We find that Pol1 and Pol3 modulate complex chromosomal rearrangements, and we confirm that Pol4 fills the gap that can form by imprecise pairing of overhanging 39 DNA ends. Our work demon- strates that multiple DNA polymerases play important roles in NHEJ. [19]. Pol b is a monomeric polymerase in vertebrates that mediates base excision repair [20,21]. Moreover, Pol1, Pol2 and Pol3 are required for HR [22–24]. Studies in mammals and yeast have provided evidence that Pol a [25], Pol e [26], Pol4 [15] and Pol m [27] play roles in NHEJ. However, much of these data were obtained through in vitro experiments, measuring religation of linear plasmids by cell extracts. Thus, it is not clear which DNA polymerases participate in NHEJ in vivo and under what circumstances. For example, DSBs on plasmids with 59-overhangs do not depend on Pol4 for their repair [16]. Conversely, chromosomal breaks due to HO endonuclease, which generates breaks with 39 overhangs, do rely on Pol4 for their repair [16]. In this paper we examine the roles of five DNA polymerases (Pol1, Pol2, Pol3, Pol4 and Rev3) in both imprecise end joining and in NHEJ-mediated chromosomal rearrangements in S. cerevisiae after a defined HO-induced DSB. These are all members of DNA polymerase families that are conserved from yeast to mammal [17]. Consistent with a previous report [15], we also observed that Pol4 is required for filling in gaps imprecise end joining. On the contrary, Pol2 is involved in deleting bases during imprecise end joining. Both Pol1 and Pol3 are required for chromosomal rearrangements, but Pol3 is not involved imprecise end joining. Our results suggest that most DNA polymerases are involved in NHEJ and each plays a distinct role in the detailed mechanisms of NHEJ. Results The In Vivo NHEJ Assay System For our in vivo NHEJ assay, a copy of the ACT1 intron was placed within the URA3 gene on chromosome V and an HO endonuclease cut site was engineered in the middle of the intron (Figure 1) [28]. A galactose inducible copy of the HO- endonuclease gene is present at the ADE3 locus on chromosome VII. Other endogenous HO cut sites, as well as the endogenous ACT1 intron, were deleted. Therefore, upon growth on galactose, the HO endonuclease creates a unique DSB in the middle of URA3 that must be repaired for cells to survive (Figure 1). Because Pol2 Is Required for NHEJ there are no other MAT related sequences in this haploid strain, the DSB cannot be repaired by the HR machinery, and most cells die. Survival is dependent on inefficient NHEJ repair pathways. Precise religation recreates the cut site, which can then be recut by the induced HO endonuclease. The most commonly observed stable repair event is imprecise NHEJ [29]. The imprecision of the end joining eliminates the endonuclease recognition sequence, but these sequence changes do not disrupt splicing because the repair occurs within a nonessential region of the intron. Repair events which interfere with the expression of URA3, however, can be identified by selecting for survivors that have become uracil auxotrophs (i.e., resistant to the drug 5-fluoro orotic acid or 5- FOA). Such repair events can include large insertions, deletions extending past the intron sequence, or chromosomal rearrange- ments such as translocations or inversions that separate the two halves of URA3 [28,30,31]. PCR amplification of the URA3::ACT1 intron::HO cut site allele, Southern blot analysis and/or sequencing of FOAR survivors allow us to distinguish such rearrangements (Figure S1). Addition of Bases during Imprecise End Joining Depends On Pol4 To determine which DNA polymerases participate in NHEJ in S. cerevisiae, we focused on five different types of DNA polymerases, including two non-essential DNA polymerases (Pol4 and Rev3) and three essential DNA polymerases (Pol1, Pol2 and Pol3). For the nonessential genes, wild-type and deletion mutants were initially grown in rich, galactose-containing, medium (YPGal) to induce HO endonuclease expression. The frequency of imprecise end joining was calculated as the ratio of colonies growing on YPGal compared to colonies growing on rich, glucose-containing medium (YPD) as described in Materials and Methods. The frequency of chromosomal rearrangements (and/or potential Figure 1. The experimental system. Structure of the URA3 allele on S. cerevisiae chromosome V used for these experiments. The position of various oligonucleotide primers (numbers and half arrows) used for PCR and sequencing are shown. The ACT1 intron placed into URA3 is normally spliced, resulting in uracil prototrophy (Ura+) and sensitivity to the drug FOAS. After creating a DSB within the engineered ACT1 intron with HO endonuclease, cells either die, are repaired in a way allowing normal splicing, or are repaired in a way that prevents splicing. The latter situation leads to a phenotype of uracil auxotrophy (Ura2) and R resistance to 5-FOA (FOA ). doi:10.1371/journal.pgen.1000060.g001 PLoS Genetics | www.plosgenetics.org 2 April 2008 | Volume 4 | Issue 4 | e1000060