Current Genetics

, Volume 64, Issue 3, pp 697–712 | Cite as

Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2

  • Michael Leshets
  • Dharanidharan Ramamurthy
  • Michael Lisby
  • Norbert Lehming
  • Ophry PinesEmail author
Original Article


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.


Fumarase DNA damage response Homologous recombination HR repair pathway Double-strand break resection Sae2 



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.

Supplementary material

294_2017_786_MOESM1_ESM.pdf (72 kb)
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 (88 kb)
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 (192 kb)
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 (44 kb)
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)


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Authors and Affiliations

  1. 1.Department of Microbiology and Molecular Genetics, IMRIC, Faculty of MedicineHebrew University of JerusalemJerusalemIsrael
  2. 2.CREATE-NUS-HUJ Program and the Department of Microbiology and Immunology, Yong Loo Lin School of MedicineNational University of SingaporeSingaporeSingapore
  3. 3.Department of BiologyUniversity of CopenhagenCopenhagen NDenmark

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