While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.

Department of Genetics Faculty of Natural Sciences Comenius University in Bratislava Bratislava Slovakia

Gregor Mendel Institute Austrian Academy of Sciences Vienna Biocenter Masaryk University Brno Czech Republic

Gregor Mendel Institute Austrian Academy of Sciences Vienna Biocenter Vienna Austria; Department of Genetics Faculty of Natural Sciences Comenius University in Bratislava Bratislava Slovakia

Zobrazit více v PubMed

Nagaria P, Robert C, Rassool FV (2013) DNA double-strand break response in stem cells: mechanisms to maintain genomic integrity. Biochim Biophys Acta 1830: 2345–2353. PubMed

Helle F (2012) Germ cell DNA-repair systems-possible tools in cancer research? Cancer Gene Ther 19: 299–302. PubMed

Riha K, McKnight TD, Griffing LR, Shippen DE (2001) Living with genome instability: plant responses to telomere dysfunction. Science 291: 1797–1800. PubMed

Doonan JH, Sablowski R (2010) Walls around tumours - why plants do not develop cancer. Nat Rev Cancer 10: 794–802. PubMed

Bray CM, West CE (2005) DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity. New Phytol 168: 511–528. PubMed

Waterworth WM, Drury GE, Bray CM, West CE (2011) Repairing breaks in the plant genome: the importance of keeping it together. New Phytol 192: 805–822. PubMed

Mannuss A, Trapp O, Puchta H (2012) Gene regulation in response to DNA damage. Biochim Biophys Acta 1819: 154–165. PubMed

Schaefer DG (2001) Gene targeting in Physcomitrella patens. Curr Opin Plant Biol 4: 143–150. PubMed

Singh SK, Roy S, Choudhury SR, Sengupta DN (2010) DNA repair and recombination in higher plants: insights from comparative genomics of Arabidopsis and rice. BMC Genomics 11: 443. PubMed PMC

Vlcek D, Sevcovicova A, Sviezena B, Galova E, Miadokova E (2008) Chlamydomonas reinhardtii: a convenient model system for the study of DNA repair in photoautotrophic eukaryotes. Curr Genet 53: 1–22. PubMed

De Muyt A, Pereira L, Vezon D, Chelysheva L, Gendrot G, et al. (2009) A high throughput genetic screen identifies new early meiotic recombination functions in Arabidopsis thaliana. PLoS Genet 5: e1000654. PubMed PMC

Timofejeva L, Skibbe DS, Lee S, Golubovskaya I, Wang R, et al. (2013) Cytological characterization and allelism testing of anther developmental mutants identified in a screen of maize male sterile lines. G3 (Bethesda) 3: 231–249. PubMed PMC

Takeda S, Tadele Z, Hofmann I, Probst AV, Angelis KJ, et al. (2004) BRU1, a novel link between responses to DNA damage and epigenetic gene silencing in Arabidopsis. Genes Dev 18: 782–793. PubMed PMC

Ulm R, Revenkova E, di Sansebastiano GP, Bechtold N, Paszkowski J (2001) Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis. Genes Dev 15: 699–709. PubMed PMC

Hefner E, Preuss SB, Britt AB (2003) Arabidopsis mutants sensitive to gamma radiation include the homologue of the human repair gene ERCC1. J Exp Bot 54: 669–680. PubMed

Gutman BL, Niyogi KK (2004) Chlamydomonas and Arabidopsis. A dynamic duo. Plant Physiol 135: 607–610. PubMed PMC

Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245–250. PubMed PMC

Molnar A, Schwach F, Studholme DJ, Thuenemann EC, Baulcombe DC (2007) miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447: 1126–1129. PubMed

Feng S, Cokus SJ, Zhang X, Chen PY, Bostick M, et al. (2010) Conservation and divergence of methylation patterning in plants and animals. Proc Natl Acad Sci U S A 107: 8689–8694. PubMed PMC

Sodeinde OA, Kindle KL (1993) Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 90: 9199–9203. PubMed PMC

Mages W, Heinrich O, Treuner G, Vlcek D, Daubnerova I, et al. (2007) Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) point mutation by recombination with a truncated nonfunctional ARG7 gene. Protist 158: 435–446. PubMed

Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 54: 1665–1669. PubMed PMC

Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph7″ gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153: 401–412. PubMed

Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87: 1228–1232. PubMed PMC

Borevitz JO, Liang D, Plouffe D, Chang HS, Zhu T, et al. (2003) Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res 13: 513–523. PubMed PMC

Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual. New Yoprk: CSHL Press.

Kazda A, Zellinger B, Rossler M, Derboven E, Kusenda B, et al. (2012) Chromosome end protection by blunt-ended telomeres. Genes Dev 26: 1703–1713. PubMed PMC

Plecenikova A, Mages W, Andresson OS, Hrossova D, Valuchova S, et al. (2013) Studies on recombination processes in two Chlamydomonas reinhardtii endogenous genes, NIT1 and ARG7. Protist 164: 570–582. PubMed

Matsuo T, Okamoto K, Onai K, Niwa Y, Shimogawara K, et al. (2008) A systematic forward genetic analysis identified components of the Chlamydomonas circadian system. Genes Dev 22: 918–930. PubMed PMC

Nelson JA, Lefebvre PA (1995) Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii. Mol Cell Biol 15: 5762–5769. PubMed PMC

Zorin B, Hegemann P, Sizova I (2005) Nuclear-gene targeting by using single-stranded DNA avoids illegitimate DNA integration in Chlamydomonas reinhardtii. Eukaryot Cell 4: 1264–1272. PubMed PMC

Zorin B, Lu Y, Sizova I, Hegemann P (2009) Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene. Gene 432: 91–96. PubMed

Yoshiyama K, Conklin PA, Huefner ND, Britt AB (2009) Suppressor of gamma response 1 (SOG1) encodes a putative transcription factor governing multiple responses to DNA damage. Proc Natl Acad Sci U S A 106: 12843–12848. PubMed PMC

Tuncay H, Findinier J, Duchene T, Cogez V, Cousin C, et al. (2013) A forward genetic approach in Chlamydomonas reinhardtii as a strategy for exploring starch catabolism. PLoS One 8: e74763. PubMed PMC

Dent RM, Haglund CM, Chin BL, Kobayashi MC, Niyogi KK (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol 137: 545–556. PubMed PMC

Davies AA, Friedberg EC, Tomkinson AE, Wood RD, West SC (1995) Role of the Rad1 and Rad10 proteins in nucleotide excision repair and recombination. J Biol Chem 270: 24638–24641. PubMed

Heitzeberg F, Chen IP, Hartung F, Orel N, Angelis KJ, et al. (2004) The Rad17 homologue of Arabidopsis is involved in the regulation of DNA damage repair and homologous recombination. Plant J 38: 954–968. PubMed

Shachar S, Ziv O, Avkin S, Adar S, Wittschieben J, et al. (2009) Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals. EMBO J 28: 383–393. PubMed PMC

Shen X, Jun S, O’Neal LE, Sonoda E, Bemark M, et al. (2006) REV3 and REV1 play major roles in recombination-independent repair of DNA interstrand cross-links mediated by monoubiquitinated proliferating cell nuclear antigen (PCNA). J Biol Chem 281: 13869–13872. PubMed

Wu F, Lin X, Okuda T, Howell SB (2004) DNA polymerase zeta regulates cisplatin cytotoxicity, mutagenicity, and the rate of development of cisplatin resistance. Cancer Res 64: 8029–8035. PubMed

Sakamoto A, Lan VT, Hase Y, Shikazono N, Matsunaga T, et al. (2003) Disruption of the AtREV3 gene causes hypersensitivity to ultraviolet B light and gamma-rays in Arabidopsis: implication of the presence of a translesion synthesis mechanism in plants. Plant Cell 15: 2042–2057. PubMed PMC

Inagaki S, Suzuki T, Ohto MA, Urawa H, Horiuchi T, et al. (2006) Arabidopsis TEBICHI, with helicase and DNA polymerase domains, is required for regulated cell division and differentiation in meristems. Plant Cell 18: 879–892. PubMed PMC

Goff JP, Shields DS, Seki M, Choi S, Epperly MW, et al. (2009) Lack of DNA polymerase theta (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation. Radiat Res 172: 165–174. PubMed PMC

Higgins GS, Prevo R, Lee YF, Helleday T, Muschel RJ, et al. (2010) A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown. Cancer Res 70: 2984–2993. PubMed PMC

Chan SH, Yu AM, McVey M (2010) Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila. PLoS Genet 6: e1001005. PubMed PMC

Mimitou EP, Symington LS (2008) Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455: 770–774. PubMed PMC

Nicolette ML, Lee K, Guo Z, Rani M, Chow JM, et al. (2010) Mre11-Rad50-Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks. Nat Struct Mol Biol 17: 1478–1485. PubMed PMC

Prinz S, Amon A, Klein F (1997) Isolation of COM1, a new gene required to complete meiotic double-strand break-induced recombination in Saccharomyces cerevisiae. Genetics 146: 781–795. PubMed PMC

Sartori AA, Lukas C, Coates J, Mistrik M, Fu S, et al. (2007) Human CtIP promotes DNA end resection. Nature 450: 509–514. PubMed PMC

Limbo O, Chahwan C, Yamada Y, de Bruin RA, Wittenberg C, et al. (2007) Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination. Mol Cell 28: 134–146. PubMed PMC

Uanschou C, Siwiec T, Pedrosa-Harand A, Kerzendorfer C, Sanchez-Moran E, et al. (2007) A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene. EMBO J 26: 5061–5070. PubMed PMC

Sarkar N, Lemaire S, Wu-Scharf D, Issakidis-Bourguet E, Cerutti H (2005) Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage. Eukaryot Cell 4: 262–273. PubMed PMC

Cenkci B, Petersen JL, Small GD (2003) REX1, a novel gene required for DNA repair. J Biol Chem 278: 22574–22577. PubMed

Debuchy R, Purton S, Rochaix JD (1989) The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J 8: 2803–2809. PubMed PMC

Purton S, Rochaix JD (1995) Characterisation of the ARG7 gene of Chlamydomonas reinhardtii and its application to nuclear transformation. Eur J Phycol 30: 141–148.

Liu J, Ren X, Yin H, Wang Y, Xia R, et al. (2010) Mutation in the catalytic subunit of DNA polymerase alpha influences transcriptional gene silencing and homologous recombination in Arabidopsis. Plant J 61: 36–45. PubMed

Barrero JM, Gonzalez-Bayon R, del Pozo JC, Ponce MR, Micol JL (2007) INCURVATA2 encodes the catalytic subunit of DNA Polymerase alpha and interacts with genes involved in chromatin-mediated cellular memory in Arabidopsis thaliana. Plant Cell 19: 2822–2838. PubMed PMC

Kirik A, Pecinka A, Wendeler E, Reiss B (2006) The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants. Plant Cell 18: 2431–2442. PubMed PMC