bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.232652; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Class-I and Class-II fumarases are a paradigm of the recruitment of 2 metabolites and metabolic enzymes for signalling of the DNA Damage 3 Response during evolution. 4 5 Yardena Silas 1, 2, Esti Singer 1, Norbert Lehming 2 and Ophry Pines 1, 2* 6 1. Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, 7 Hebrew University, Jerusalem, Israel 8 2. CREATE‑NUS‑HUJ Program and the Department of Microbiology and Immunology, 9 Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 10 Singapore. 11 12 [email protected] 13 [email protected] 14 [email protected] 15 [email protected] 16 17 18 19 20 21 22 23 24 25 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.232652; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 27 Abstract 28 Class-II fumarase (Fumarate Hydratase, FH) and its metabolic intermediates are essential 29 components in the DNA damage response (DDR) in eukaryotic cells (human and yeast) and 30 in the prokaryote Bacillus subtilis. Strikingly, here we show, in Escherichia coli, which harbors 31 three fumarase genes; Class-I fumA and fumB and Class-II fumC, a variation of the distribution 32 of function so far demonstrated (TCA cycle and DDR). Contrary to previous reports, E. coli 33 Class-II fumarase participates naturally in respiration and the Class-I fumarases in the DDR. 34 Contrary to other organisms, in E. coli, alpha-ketoglutarate (α-KG) is the organic acid that 35 complements DNA damage sensitivity of fum null mutants. We show inhibition of the α-KG- 36 dependent DNA damage repair enzyme, AlkB, by fumarate and succinate, and a global effect 37 of fumarase absence on transcription. Together these results show an adaptable metabolic 38 signalling of the DDR during evolution regardless of the enzyme Class preforming the DDR 39 related function. 40 41 Introduction 42 Fumarase (fumarate hydratase, FH) is an enzyme that participates in the tricarboxylic acid 43 (TCA) cycle, there, it catalyzes the reversible hydration of fumarate to L-malate (Woods, 44 Schwartzbach, & Guest, 1988). In eukaryotes, in addition to its mitochondrial localization, a 45 common theme conserved from yeast to humans is the existence of a cytosolic form of 46 fumarase (Akiba, Hiraga, & Tuboi, 1984; Tuboi, Suzuki, Sato, & Yoshida, 1990). The dual 47 distribution of fumarase between the mitochondria and the cytosol is a universal trait, but 48 intriguingly, the mechanism by which dual distribution occurs does not appear to be conserved 49 (Dik, Naamati, Asraf, Lehming, & Pines, 2016; Karniely, Regev-Rudzki, & Pines, 2006; 50 Pracharoenwattana et al., 2010; Regev-Rudzki, Yogev, & Pines, 2008; Sass, Karniely, & Pines, 51 2003). We discovered that in eukaryotes the cytosolic form of this TCA enzyme was shown to 52 have an unexpected function in the DNA damage response (DDR), and specifically, a role in 53 recovery from DNA double strand breaks (DSBs) (Yogev et al., 2010). It appears that 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.232652; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 54 metabolic signaling in these organisms is achieved via the fumarase organic acid substrate 55 fumarate (Jiang et al., 2018; Leshets, Ramamurthy, Lisby, Lehming, & Pines, 2018). A recent 56 study by Singer et al. (Singer, Silas, Ben-Yehuda, & Pines, 2017) shows that the Gram- 57 positive Bacillus subtilis bacterial fumarase, has a role in the TCA cycle, yet also participates 58 in the DDR. Fumarase dependent signaling of the DDR is achieved by its metabolic product, 59 L-malate, which affects RecN (the first protein recruited to DNA damage sites) at the level of 60 expression levels and localization. That study indicated that the dual function of this enzyme 61 predated its dual distribution and, according to our model, was the driving force for the 62 evolution of dual targeting in eukaryotes (Singer et al., 2017). 63 An intriguing variation to the themes above, is the fact that there are two distinct classes of 64 fumarase. In the organisms mentioned above (Sacchromyces cerevisiae, human, B. subtilis) 65 the fumarase that participates in both the TCA cycle and in DNA repair, belongs to the Class- 66 II fumarase. In contrast, the Class-I fumarase, which bears no sequence or structural similarity 67 to Class-II fumarases, is predominantly found in prokaryotes and in early evolutionary 68 divergents in the eukaryotic kingdom (protozoa and invertebrates) (Woods et al., 1988). The 69 model organism that we have employed in this study is the Gram-negative bacterium E. coli 70 which harbors three fumarase genes; Class-I, fumA and fumB and Class-II, fumC. FumA and 71 FumB are homologous proteins, sharing 90% amino acid sequence identity. Both FumA and 72 FumB kinetic parameters are very similar with respect to the natural substrates L-malate and 73 fumarate (van Vugt-Lussenburg, van der Weel, Hagen, & Hagedoorn, 2013). FumC is 74 homologous to eukaryotic and B. subtilis fumarases, sharing 64% amino acid sequence 75 identity with eukaryotic and B. subtilis fumarases. 76 Employing the E. coli model, we ask whether Class-I and/or Class-II fumarases are involved 77 in the DDR and/or the TCA cycle and what is the distribution of functions between these two 78 Classes of fumarases in this organism. Here we show that FumA and FumB are participants 79 of the DDR in E. coli, while FumC naturally participates in respiration. FumA and FumB 80 participation in the DDR is based on a fascinating interplay of TCA cycle Intermediates, alpha- 81 ketoglutarate (α-KG), fumarate and succinate. In E. coli the absence of fumarase is shown to 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.232652; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 82 affect the transcription of many genes following DNA damage induction, including many DNA 83 repair genes. Specifically, we show that the target of alpha-ketoglutarate is the α-KG- 84 dependent DNA repair enzyme AlkB demethylase which is required for successful repair of 85 DNA damage and which is also modulated by fumarate and succinate. 86 87 Results 88 fumA and fumB but not fumC E. coli strains, are sensitive to DNA damage 89 induction. 90 In order to examine a possible role for E. coli fumarases in the DNA damage response, we 91 induced DNA damage in E. coli strains lacking each of the fumarase genes, by exposing them 92 to treatment with Ionizing radiation (IR, Fig. 1A) or methyl methanesulfonate (Fig. 1B, MMS, 93 0.35% [v/v] for 30 or 45 minutes). Figures 1A and 1B (compare rows 2 and 3 in each panel to 94 row 1) demonstrate that compared to the control (WT), strains lacking fumA or fumB are very 95 sensitive to DNA damage (IR or 0.35% [v/v] MMS for 45 minutes), while the strain lacking 96 fumC seems unaffected by the DNA damage induction (row 4). These results suggest that 97 FumA and FumB play a role in the DNA damage response, while FumC has no observable 98 role in this process. While the levels of FumA exhibit a very insignificant change in protein 99 levels upon treatment with MMS (Fig 1c top panel), the levels of FumC increase (Fig 1C 100 second panel), thereby revealing a more complex picture of regulation. Intriguingly, the double 101 mutant (Fig 1D, middle panel, fumAB, Row 5) is resistant to DNA damage induction, either 102 IR or MMS treatment and shows a phenotype resembling the WT strain (Row 1), when 103 compared to single fumA and fumB deletion strains (fumA, fumB, rows 2 and 3) or a triple 104 mutant lacking all three fumarase genes (fumACB, row 6) which are sensitive to IR and MMS. 105 One possible explanation for this phenomenon is that in the absence of FumA and FumB, 106 FumC expression, which is much lower at basal levels (i.e. no MMS treatment) increases to 107 undertake both roles of respiration and participation in the DDR (Fig 1E). Worth mentioning 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.232652; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 108 here and referred to in the next section, is that complementation of the DNA damage sensitivity 109 can be achieved by the genes in trans (Fig 3).