Abstract
One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the main DSB repair pathways. Fumarase is a mitochondrial enzyme which functions in the tricarboxylic acid cycle. Intriguingly, the enzyme can be readily detected in the cytosolic compartment of all organisms examined, and we have shown that cytosolic fumarase participates in the DNA damage response towards DSBs. In human cells, fumarase was shown to be involved in NHEJ, but it is still unclear whether fumarase is also important for the HR pathway. Here we show that the depletion of cytosolic fumarase in yeast prolongs the presence of Mre11 at the DSBs, and decreases the kinetics of repair by the HR pathway. Overexpression of Sae2 endonuclease reduced the DSB sensitivity of the cytosolic fumarase depleted yeast, suggesting that Sae2 and fumarase functionally interact. Our results also suggest that Sae2 and cytosolic fumarase physically interact in vivo. Sae2 has been shown to be important for the DSB resection process, which is essential for the repair of DSBs by the HR pathway. Depletion of cytosolic fumarase inhibited DSB resection, while the overexpression of cytosolic fumarase or Sae2 restored resection. Together with our finding that cytosolic fumarase depletion reduces Sae2 cellular amounts, our results suggest that cytosolic fumarase is important for the DSB resection process by regulating Sae2 levels.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adamczyk J, Deregowska A, Panek A, Golec E, Lewinska A, Wnuk M (2016) Affected chromosome homeostasis and genomic instability of clonal yeast cultures. Curr Genet 62:405–418. https://doi.org/10.1007/s00294-015-0537-3
Akiba T, Hiraga K, Tuboi S (1984) Intracellular distribution of fumarase in various animals. J Biochem 96:189–195
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2004) Molecular biology of the cell, 5th edn. WH Freeman
An J et al (2010) DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression. BMC Mol Biol 11:18. https://doi.org/10.1186/1471-2199-11-18
Anand R, Ranjha L, Cannavo E, Cejka P (2016) Phosphorylated CtIP functions as a co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end. Resect Mol Cell 64:940–950. https://doi.org/10.1016/j.molcel.2016.10.017
Ang K et al. (2012) Mediator acts upstream of the transcriptional activator Gal4. PLoS Biol 10:e1001290. https://doi.org/10.1371/journal.pbio.1001290
Aylon Y, Kupiec M (2004) DSB repair: the yeast paradigm DNA Repair. (Amst) 3:797–815. https://doi.org/10.1016/j.dnarep.2004.04.013
Barker KT et al (2002) Low frequency of somatic mutations in the FH/multiple cutaneous leiomyomatosis gene in sporadic leiomyosarcomas and uterine leiomyomas. Br J Cancer 87:446–448. https://doi.org/10.1038/sj.bjc.660502
Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F, Cullin C (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330
Bermudez-Lopez M, Aragon L (2017) Smc5/6 complex regulates Sgs1 recombination functions. Curr Genet 63:381–388. https://doi.org/10.1007/s00294-016-0648-5
Burak E, Yogev O, Sheffer S, Schueler-Furman O, Pines O (2013) Evolving dual targeting of a prokaryotic protein in yeast. Mol Biol Evol 30:1563–1573. https://doi.org/10.1093/molbev/mst039
Cannavo E, Cejka P (2014) Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect. DNA Breaks Nat 514:122–125. https://doi.org/10.1038/nature13771
Chen H, Lisby M, Symington LS (2013) RPA coordinates DNA end resection and prevents formation of. DNA Hairpins Mol Cell 50:589–600. https://doi.org/10.1016/j.molcel.2013.04.032
Chung WH, Zhu Z, Papusha A, Malkova A, Ira G (2010) Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet 6:e1000948. https://doi.org/10.1371/journal.pgen.1000948
Clerici M, Mantiero D, Lucchini G, Longhese MP (2005) The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends. J Biol Chem 280:38631–38638. https://doi.org/10.1074/jbc.M508339200
Clerici M, Mantiero D, Lucchini G, Longhese MP (2006) The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling. EMBO Rep 7:212–218. https://doi.org/10.1038/sj.embor.7400593
Clerici M, Mantiero D, Guerini I, Lucchini G, Longhese MP (2008) The Yku70-Yku80 complex contributes to regulate double-strand break processing and checkpoint activation during the cell cycle. EMBO Rep 9:810–818. https://doi.org/10.1038/embor.2008.121
Cross FR (1997) ‘Marker swap’ plasmids: convenient tools for budding yeast molecular genetics. Yeast 13:647–653. https://doi.org/10.1002/(SICI)1097-0061(19970615)13:7<;647::AID-YEA115>;3.0.CO;2-#
Daley JM, Wilson TE (2005) Rejoining of DNA double-strand breaks as a function of overhang length. Mol Cell Biol 25:896–906. https://doi.org/10.1128/MCB.25.3.896-906.2005
Davis AP, Symington LS (2003) The Rad52-Rad59 complex interacts with Rad51 and replication protein A DNA Repair. (Amst) 2:1127–1134
Duina AA, Miller ME, Keeney JB (2014) Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae. Model Syst Genet 197:33–48. https://doi.org/10.1534/genetics.114.163188
Durdikova K, Chovanec M (2017) Regulation of non-homologous end joining via post-translational modifications of components of the ligation step. Curr Genet 63:591–605. https://doi.org/10.1007/s00294-016-0670-7
Eckert-Boulet N, Rothstein R, Lisby M (2011) Cell biology of homologous recombination in yeast. Methods Mol Biol 745:523–536. https://doi.org/10.1007/978-1-61779-129-1_30
Edwards YH, Hopkinson DA (1979) Further characterization of the human fumarase variant. Ann Human Genet 43:FH 2–1 103–108
Ensminger M, Iloff L, Ebel C, Nikolova T, Kaina B, Lbrich M (2014) DNA breaks and chromosomal aberrations arise when replication meets base excision repair. J Cell Biol 206:29–43. https://doi.org/10.1083/jcb.201312078
Evans M, Griffiths H, Lunec J (1997) Reactive oxygen species and their cytotoxic mechanisms. Adv Mol Cell Biol 20:25–73
Ferrari M et al (2015) Functional interplay between the 53BP1-ortholog Rad9 and the Mre11 complex regulates resection, end-tethering and repair of a double-strand break. PLoS genetics. 11:e1004928 https://doi.org/10.1371/journal.pgen.1004928
Frank-Vaillant M, Marcand S (2002) Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination. Mol Cell 10:1189–1199
Funakoshi M, Hochstrasser M (2009) Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae. Yeast 26:185–192. https://doi.org/10.1002/yea.1658
Game JC, Mortimer RK (1974) A genetic study of X-ray sensitive mutants in yeast. Mutat Res 24:281–292
Garcia V, Phelps SE, Gray S, Neale MJ (2011) Bidirectional resection of DNA double-strand breaks by Mre11 and Exo. Nature 479(1):241–244. https://doi.org/10.1038/nature10515
Gardie B et al (2011) Novel FH mutations in families with hereditary leiomyomatosis and renal cell cancer (HLRCC) and patients with isolated type 2 papillary renal cell carcinoma. J Med Genet 48:226–234. https://doi.org/10.1136/jmg.2010.085068
Haber JE, Ray BL, Kolb JM, White CI (1993) Rapid kinetics of mismatch repair of heteroduplex DNA that is formed during recombination in yeast. Proc Natl Acad Sci USA 90:3363–3367
Hamilton NK, Maizels N (2010) MRE11 function in response to topoisomerase poisons is independent of its function in double-strand break repair in Saccharomyces cerevisiae. PLoS One 5:e15387. https://doi.org/10.1371/journal.pone.0015387
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674. https://doi.org/10.1016/j.cell.2011.02.013
Hays SL, Firmenich AA, Berg P (1995) Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc Natl Acad Sci USA 92:6925–6929
Herskowitz I, Jensen RE (1991) Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol 194:132–146
Hicks J, Strathern JN, Klar AJ (1979) Transposable mating type genes in Saccharomyces cerevisiae. Nature 282:478–473
Hopfner KP, Karcher A, Craig L, Woo TT, Carney JP, Tainer JA (2001) Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Cell 105:473–485
Ira G et al (2004) DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431:1011–1017. https://doi.org/10.1038/nature02964
Isaacs JS et al (2005) HIF overexpression correlates with biallelic loss of fumarate hydratase in renal cancer: novel role of fumarate in regulation of HIF stability. Cancer Cell 8:143–153. https://doi.org/10.1016/j.ccr.2005.06.017
Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461:1071–1078. https://doi.org/10.1038/nature08467
Jiang Y et al (2015) Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation. Nat Cell Biol 17:1158–1168. https://doi.org/10.1038/ncb3209
Kim HS, Vijayakumar S, Reger M, Harrison JC, Haber JE, Weil C, Petrini JH (2008) Functional interactions between Sae2 and the Mre11 complex. Genetics 178:711–723. https://doi.org/10.1534/genetics.107.081331
Kiuru M et al (2001) Familial cutaneous leiomyomatosis is a two-hit condition associated with renal cell cancer of characteristic histopathology. Am J Pathol 159:825–829. https://doi.org/10.1016/S0002-9440(10)61757-9
Kiuru M et al (2002) Few FH mutations in sporadic counterparts of tumor types observed in hereditary leiomyomatosis and renal cell cancer families. Cancer Res 62:4554–4557
Kobayashi K, Tuboi S (1983) End group analysis of the cytosolic and mitochondrial fumarases from rat liver. J Biochem 94:707–713
Kramer KM, Brock JA, Bloom K, Moore JK, Haber JE (1994) Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events. Mol Cell Biol 14:1293–1301
Laser H, Bongards C, Schuller J, Heck S, Johnsson N, Lehming N (2000) A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter. Proc Natl Acad Sci USA 97:13732–13737. https://doi.org/10.1073/pnas.250400997
Launonen V et al (2001) Inherited susceptibility to uterine leiomyomas and renal cell cancer. Proc Natl Acad Sci USA 98:3387–3392. https://doi.org/10.1073/pnas.051633798
Lee SE, Moore JK, Holmes A, Umezu K, Kolodner RD, Haber JE (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after. DNA Damage Cell 94:399–409
Lee SJ, Schwartz MF, Duong JK, Stern DF (2003) Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling. Mol Cell Biol 23:6300–6314
Lehming N (2002) Analysis of protein-protein proximities using the split-ubiquitin system. Brief Funct Genom Proteom 1:230–238
Lehtonen R et al (2004) Biallelic inactivation of fumarate hydratase (FH) occurs in nonsyndromic uterine leiomyomas but is rare in other tumors. Am J Pathol 164:17–22. https://doi.org/10.1016/S0002-9440(10)63091-X
Lengauer C, Kinzler KW, Vogelstein B (1998) Genetic instabilities in human cancers. Nature 396:643–649. https://doi.org/10.1038/25292
Lewis LK, Resnick MA (2000) Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae. Mutat Res 451:71–89
Li X, Heyer WD (2009) RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae. Nucleic Acids Res 37:638–646. https://doi.org/10.1093/nar/gkn980
Lindahl T, Barnes DE (2000) Repair of endogenous DNA damage. Cold Spring Harbor Symp Quant Biol 65:127–133
Lisby M, Barlow JH, Burgess RC, Rothstein R (2004) Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and. repair. Proteins Cell 118:699–713. https://doi.org/10.1016/j.cell.2004.08.015
Majka J, Binz SK, Wold MS, Burgers PM (2006) Replication protein A directs loading of the DNA damage checkpoint clamp to 5′-DNA junctions. J Biol Chem 281:27855–27861. https://doi.org/10.1074/jbc.M605176200
Mann PJ, Woolf B (1930) The action of salts on fumarase. I. Biochem J 24:427–434
Milne GT, Weaver DT (1993) Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52. Genes Dev 7:1755–1765
Mimitou EP, Symington LS (2008) Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455:770–774. https://doi.org/10.1038/nature07312
Mimitou EP, Symington LS (2010) Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2. EMBO J 29:3358–3369. https://doi.org/10.1038/emboj.2010.193
Miyazaki T, Bressan DA, Shinohara M, Haber JE, Shinohara A (2004) In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J 23:939–949. https://doi.org/10.1038/sj.emboj.7600091
Moore JK, Haber JE (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 16:2164–2173
Moreau S, Ferguson JR, Symington LS (1999) The nuclease activity of Mre11 is required for meiosis but not for mating type switching, end joining, or telomere maintenance. Mol Cell Biol 19:556–566
Nakada D, Matsumoto K, Sugimoto K (2003) ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev 17:1957–1962. https://doi.org/10.1101/gad.1099003
Nickoloff JA, Singer JD, Hoekstra MF, Heffron F (1989) Double-strand breaks stimulate alternative mechanisms of recombination repair. J Mol Biol 207:527–541
O’Driscoll M (2012) Diseases associated with defective responses to DNA damage. Cold Spring Harbor Perspect Biol 4. https://doi.org/10.1101/cshperspect.a012773
O’Hare MC, Doonan S (1985) Purification and structural comparisons of the cytosolic and mitochondrial isoenzymes of fumarase from pig liver. Biochim Biophys Acta 827:127–134
Pellicioli A et al (1999) Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase. EMBO J 18:6561–6572. https://doi.org/10.1093/emboj/18.22.6561
Pollard PJ et al (2005) Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Human Mol Genet 14:2231–2239. https://doi.org/10.1093/hmg/ddi227
Rattray AJ, McGill CB, Shafer BK, Strathern JN (2001) Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM. Genetics 158(1):109–122
Reed WB, Walker R, Horowitz R (1973) Cutaneous leiomyomata with uterine leiomyomata. Acta Dermato-Venereol 53:409–416
Regev-Rudzki N, Karniely S, Ben-Haim NN, Pines O (2005) Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing. Mol Biol Cell 16:4163–4171. https://doi.org/10.1091/mbc.E04-11-1028
Reid RJ, Lisby M, Rothstein R (2002) Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated. PCR Methods Enzymol 350:258–277
Richardson C, Jasin M (2000) Frequent chromosomal translocations induced by DNA double-strand breaks. Nature 405:697–700. https://doi.org/10.1038/35015097
Robert T et al (2011) HDACs link the DNA damage response processing of double-strand breaks autophagy. Nature 471:74–79. https://doi.org/10.1038/nature09803
Saintigny Y, Delacote F, Vares G, Petitot F, Lambert S, Averbeck D, Lopez BS (2001) Characterization of homologous recombination induced by replication inhibition in mammalian cells. EMBO J 20:3861–3870. https://doi.org/10.1093/emboj/20.14.3861
Sanchez Y, Desany BA, Jones WJ, Liu Q, Wang B, Elledge SJ (1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271:357–360
Sarto-Jackson I, Tomaska L (2016) How to bake a brain: yeast as a model neuron. Curr Genet 62:347–370. https://doi.org/10.1007/s00294-015-0554-2
Sass E, Blachinsky E, Karniely S, Pines O (2001) Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini. J Biol Chem 276:46111–46117. https://doi.org/10.1074/jbc.M106061200
Sass E, Karniely S, Pines O (2003) Folding of fumarase during mitochondrial import determines its dual targeting in yeast. J Biol Chem 278:45109–45116. https://doi.org/10.1074/jbc.M302344200
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Selak MA et al (2005) Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell 7:77–85. https://doi.org/10.1016/j.ccr.2004.11.022
Shiloh Y, Lehmann AR (2004) Maintaining integrity. Nat Cell Biol 6:923–928. https://doi.org/10.1038/ncb1004-923
Shroff R et al (2004) Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. CB 14:1703–1711. https://doi.org/10.1016/j.cub.2004.09.047
Song B, Sung P (2000) Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange. J Biol Chem 275:15895–15904. https://doi.org/10.1074/jbc.M910244199
Stein I, Peleg Y, Even-Ram S, Pines O (1994) The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae. Mol Cell Biol 14:4770–4778
Stiff T, O’Driscoll M, Rief N, Iwabuchi K, Lobrich M, Jeggo PA (2004) ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res 64:2390–2396
Sugawara N, Wang X, Haber JE (2003) In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol Cell 12:209–219
Sun Z, Fay DS, Marini F, Foiani M, Stern DF (1996) Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways. Genes Dev 10:395–406
Sung P (1997) Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. J Biol Chem 272:28194–28197
Toh GW et al (2006) Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. DNA Repair. (Amst) 5:693–703. https://doi.org/10.1016/j.dnarep.2006.03.005
Tolley E, Craig I (1975) Presence of two forms of fumarase (fumarate hydratase E.C. 4.2.1.2) in mammalian cells: immunological characterization and genetic analysis in somatic cell hybrids. Confirmation of the assignment of a gene necessary for the enzyme expression to human chromosome 1. Biochem Genet 13:867–883
Tomlinson IP et al (2002) Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 30:406–410. https://doi.org/10.1038/ng849
Trujillo KM, Roh DH, Chen L, Van Komen S, Tomkinson A, Sung P (2003) Yeast xrs2 binds DNA and helps target rad50 and mre11 to DNA ends. J Biol Chem 278:48957–48964. https://doi.org/10.1074/jbc.M309877200
Tsabar M et al (2015) Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2. Nucleic Acids Res 43:6889–6901. https://doi.org/10.1093/nar/gkv520
Usui T, Ogawa H, Petrini JH (2001) A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol Cell 7:1255–1266
van Gent DC, Hoeijmakers JH, Kanaar R (2001) Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet 2:196–206. https://doi.org/10.1038/35056049
Vanharanta S et al (2006) Distinct expression profile in fumarate-hydratase-deficient uterine fibroids. Human Mol Genet 15:97–103. https://doi.org/10.1093/hmg/ddi431
Vaze MB et al (2002) Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol Cell 10:373–385
Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808
Wang X, Haber JE (2004) Role of Saccharomyces single-stranded DNA-binding protein RPA in the strand invasion step of double-strand break repair. PLoS Biol 2:E21. https://doi.org/10.1371/journal.pbio.0020021
White CI, Haber JE (1990) Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J 9:663–673
Winzeler EA et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion parallel analysis. Science 285:901–906
Woods SA, Schwartzbach SD, Guest JR (1988) Two biochemically distinct classes of fumarase in Escherichia coli. Biochim Biophys Acta 954:14–26
Wu M, Tzagoloff A (1987) Mitochondrial and cytoplasmic fumarases in Saccharomyces cerevisiae are encoded by a single nuclear gene FUM1. J Biol Chem 262:12275–12282
Yogev O, Yogev O, Singer E, Shaulian E, Goldberg M, Fox TD, Pines O (2010) Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response. PLoS Biol 8:e1000328. https://doi.org/10.1371/journal.pbio.1000328
Zhang Y, Shim EY, Davis M, Lee SE (2009) Regulation of repair choice: Cdk1 suppresses recruitment of end joining factors at DNA breaks. DNA Repair. (Amst) 8:1235–1241. https://doi.org/10.1016/j.dnarep.2009.07.007
Zhu Z, Chung WH, Shim EY, Lee SE, Ira G (2008) Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134:981–994. https://doi.org/10.1016/j.cell.2008.08.037
Zierhut C, Diffley JF (2008) Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J 27:1875–1885. https://doi.org/10.1038/emboj.2008.111
Acknowledgements
We thank Sheera Adar for critical reading of the manuscript. This work was supported by grants to O. Pines from the Israel Science Foundation (ISF) and the German Israeli Project Cooperation (DIP). N. Lehming and O. Pines were supported by The CREATE Project of the National Research Foundation of Singapore. M. Lisby was supported by the Danish Council for Independent Research and the Villum Foundation.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by M. Kupiec.
Electronic supplementary material
Below is the link to the electronic supplementary material.
294_2017_786_MOESM1_ESM.pdf
Fig S1 (A) Logarithmic cultures of the indicated strains in which Mre11 was C-terminally fused to YFP within the genome, were visualized using fluorescence microscopy. The white arrows indicate nuclear foci formed by Mre11. (B,C) Logarithmic cultures of the strains described in Fig 1A were exposed to 0.03%(v/v) methyl methanesulfonate (MMS) for one hour and washed (Wash). Samples were collected at indicated time points and analyzed using fluorescence microscopy to determine the percentage of cells containing at least one nuclear focus. Cells examined: (B) n=2268, (C) n=5855. (D) Calculation of colony-forming units (CFU). CFU/ml values were normalized to the values obtained from the strains grown on control media, and presented as values relative to those of the WT strain. The experiments described in Fig 2A were quantified to determine the CFU/ml of the indicated strains. * for p=2.98x10-3, ** for p=2.18x10-3, error bars SEM. (E) Logarithmic cultures of the indicated strains were treated with 800mM hydroxyurea (HU). At the indicated time points, samples were collected, and the transcript levels of yKu70 were determined by quantitative PCR. Error barsindicate SD. (F) Representation of the Western blot regions quantified in Fig 2C. Nonphospho-Rad53 is indicated by a red box, phospho-Rad53 species are indicated by a blue dashed box (PDF 72 KB)
294_2017_786_MOESM2_ESM.pdf
Fig S2 (A) The transcript levels of Tel1 and Mec1 were determined as described in supplementary Fig S1E. (B) Calculation of colony-forming units (CFU). CFU/ml values were normalized to the values obtained from the strains grown on control media, and presented as values relatively to those of the WT strain. The experiments described in Fig 3B were quantified to determine the CFU/ml of the indicated strains. * for p=1.80x10-2, ** for p=2.03x10-2, error bars SEM.. (C) Overnight cultures of the indicated yeast strains were diluted to OD600=0.5 and incubated for 90 minutes at 300C. The cultures were then incubated for one hour with or without 0.2%(v/v) methyl methanesulfonate (MMS), serially diluted and plated on SC-Dex plates. (D) The indicated strains were prepared for the experiment as in C, then serially diluted and plated on SC-Dex with or without 200mM HU. (E) The experiment was conducted as in D. The indicated strains were plated on SC-Gal plates (PDF 87 KB)
294_2017_786_MOESM3_ESM.pdf
Fig S3 Protein fusions of Sae2 and fumarase are functional. (A) BY4741 Δsae2 cells expressing Sae2 fused to HA3-H10 (row 1) or Cub-RUra3 (row 4) under the SAE2 promoter and BY4741 Δsae2 cells harboring empty YCplac33 vector (row 3), were serially diluted and spotted onto plates with or without 300mM HU. (B) Wild type (BY4743ΔW; line 1) or Δfum1 (BY4743ΔWΔFUM1; line 2) cells expressing fumarase fused to Nub under the control of its endogenous promoter (lines 3 to 8) or the ADH1 promoter (lines 9 to 14), were grown to mid-log phase and induced (+HU) or not induced (-HU) with 400mM HU for two hours. Fumarase protein levels were determined by western blot analysis using anti-fumarase antibody. An anti-Pgk1 antibody was used as a loading control. (C) Serial dilutions of cells of the indicated genotypes were spotted onto plates containing or not containing 300mM HU (PDF 192 KB)
294_2017_786_MOESM4_ESM.pdf
Fig S4 The presence of cytosolic fumarase is important for the protein level of Sae2, but not for its transcript abundance. (A) Logarithmic cultures of the indicated strains were treated with 800mM HU. At the indicated time points samples were collected, and the transcript level of SAE2 was determined by quantitative PCR. Error bars SD. (B) Logarithmic cultures of WT and FumM strains, in which 3xFLAG tag was inserted at the 3’ end of SAE2 on chromosome VII, were treated with 350mM HU. At the indicated time points samples were analyzed by western blot, using anti-FLAG antibody. Aco1 was used as a loading control (control). (C) Densitometric quantification of the experiments in B. * for p=1.53x10-3, ** for p=3.67x10-4, error bars SEM (PDF 44 KB)
Rights and permissions
About this article
Cite this article
Leshets, M., Ramamurthy, D., Lisby, M. et al. Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2. Curr Genet 64, 697–712 (2018). https://doi.org/10.1007/s00294-017-0786-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-017-0786-4