Proofreading Activity of DNA Polymerase Pol2 Mediates 39-End Processing during Nonhomologous End Joining in Yeast Shun-Fu Tseng 1 , Abram Gabriel2, Shu-Chun Teng1,3* 1 Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan, 2 Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, New Jersey, United States of America, 3 Institute of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Abstract Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 39 DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 39 DNA ends, Pol2 is important for the recession of 39 flaps that can form during imprecise pairing. Indeed, a mutation in the 39-59 exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 39 flaps. Thus, Pol2 performs a key 39 end-processing step in NHEJ. Citation: Tseng S-F, Gabriel A, Teng S-C (2008) Proofreading Activity of DNA Polymerase Pol2 Mediates 39-End Processing during Nonhomologous End Joining in Yeast. PLoS Genet 4(4): e1000060. doi:10.1371/journal.pgen.1000060 Editor: James E. Haber, Brandeis University, United States of America Received August 28, 2007; Accepted March 26, 2008; Published April 25, 2008 Copyright: 2008 Tseng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Science Council (NRPGM-95-3112-B-002-023) and National Health Research Institute of Taiwan (NHRI-EX95-9328SI). Competing Interests: The authors have declared that no competing interests exist. * E-mail: shuchunteng@ntu.edu.tw Introduction DNA DSBs result from disruption of the phosphodiester backbone on both strands of a DNA double helix. They are induced by ionizing radiation and chemicals, including anticancer drugs, or can arise spontaneously during DNA replication [1–3]. Furthermore, DSBs occur normally as intermediates in V(D)J recombination, the process that helps to generate the vast range of antigen-binding sites of antibody and T-cell receptor proteins during lymphoid-cell development [4]. DNA DSBs are critical lesions that, if unrepaired or misrepaired, may be lethal for a cell or help in its malignant transformation. DSBs can be repaired either by homologous recombination (HR) or by NHEJ [1,3,5–8]. While the former process is generally error- free, the latter process is potentially error-prone. DSB repair by HR requires extensive regions of sequence homology between donor and recipient DNA strands. In NHEJ the DNA ends are joined with little or no base pairing at the junction [9]. These repair mechanisms are evolutionarily conserved, but contribute unequally to overall DSB repair in different organisms. In mammals, DSBs are primarily repaired by NHEJ, while in yeast HR dominates. The budding yeast S. cerevisiae is the most intensely studied model system for DSB DNA repair. This organism has a classical NHEJ pathway that depends on Ku and DNA ligase IV, as well as Rad50, Mre11 and Xrs2, three proteins that have endo- and exonuclease activities [10,11]. Agents that cause DSBs often create damaged or non- complementary bases at the break termini [12,13]. In these circumstances, simple religation cannot occur, and additional factors must be used to process the DNA breaks to create suitable 59 and 39 ends for ligation. A nuclease, termed Artemis, has been shown to be important for mammalian NHEJ [14]. Purified Artemis protein possesses single-strand-specific 59 to 39 exonucle- ase activity. In conjunction with the DNA-dependent protein kinase (DNA-PK), Artemis has both 59 to 39 and 39 to 59 exonuclease activities [14]. S. cerevisiae lacks both Artemis and the catalytic component of DNA-PK, and the nuclease(s) and polymerase(s) involved in the yeast NHEJ pathway are not well understood. The Pol X family of DNA polymerases has been implicated in NHEJ, since Pol4, the only Pol X family member in yeast, is required for gap filling in some end configurations [15]. Pol X polymerases appear to be required for NHEJ only when gaps must be filled, indicating that they are not part of the core NHEJ complex [15,16]. At least six nuclear DNA polymerases have been described in eukaryotic cells that participate in DNA replication and/or repair [17]. Pol1 (designated Pol a or CDC17 in yeast), Pol2 (Pol e), and Pol3 (Pol d or CDC2) together catalyze the essential functions of DNA replication. Pol2 and Pol3 are also involved in certain DNA repair events, notably nucleotide excision repair [18]. Rev3 (Pol j) and Rad30 (Pol g) mediate translesion bypass synthesis in yeast PLoS Genetics | www.plosgenetics.org 1 April 2008 | Volume 4 | Issue 4 | e1000060