Bacterial fumarase and L-malic acid are evolutionary ancient components of the DNA damage response
- IMRIC, Faculty of Medicine, Hebrew University, Israel
- National University of Singapore, Sinapore
Figures
Figure 1 with 3 supplements
Fumarase is required for an efficient DNA damage response in Bacillus subtilis.
(A) Bacillus subtilis wild type (Bs WT) and Δfum (=ΔcitG) strains were untreated (control) or exposed to ionized radiation (150 Gy) and then serially diluted onto LB plates or minimal S7 medium …
Figure 1—figure supplement 1
Expression of B. subtilis and yeast fumarases.
(A) B. subtilis wild type (Bs WT), Δfum and Δfum+Fum-bc strains were grown to logarithmic phase. The levels of fumarase in the cells were determined by western blot analysis. The Polyclonal …
Figure 1—figure supplement 2
Expression of B. subtilis and yeast fumarases.
(A) S. cerevisiae wild type (Sc WT) and Δfum1 strains harboring the indicated plasmids were grown on dextrose (DEX). After o.n growth the cells were grown on galactose (GAL) SD media to express the …
Figure 1—figure supplement 3
Subcellular fractionation of yeast cells.
Subcellular fractionation of yeast cells expressing Fum-bc. The total (T), cytosol (C), and mitochondria (M) fractions were analyzed by western blotting using the indicated antibodies. Controls: HK …
Figure 2 with 2 supplements
Fumarase is co-localized with the DNA after induction of DNA damage.
(A) B. subtilis was grown to logarithmic phase, MMS was added to final concentration of 0.07%(v/v) for 60 min. The levels of fumarase in the cells were determined at the indicated times by western …
Figure 2—figure supplement 1
TCA cycle enzymes.
(A) B. subtilis was grown to logarithmic phase, MMS was added to final concentration of 0.07%(v/v) for 60 min. The levels of citZ and ICDH in the cells were determined at the indicated times by …
Figure 2—figure supplement 2
Enzymatic activity of B. subtilis fumarase fused to GFP.
B. subtilis cells were grown to logarithmic phase, harvested and resuspended in PBS. The cells were lysed and centrifuged to obtain the supernatant which was assayed for fumarase activity at 250 nm …
Figure 3 with 4 supplements
Fumarase enzymatic activity and L-malic acid are required for the DNA damage protective function.
(A) B. subtilis wild type, Δfum and mutant H186N fumarase strains were grown to logarithmic phase. MMS was added to a final concentration of 0.07% (v/v) for 45 min, the cells were washed and …
Figure 3—figure supplement 1
Expression of mutant fumarases.
(A) B. subtilis wild type (WT), Δfum, H127R and H186N strains were grown on LB media. Total cell extracts were prepared and subjected to western blotting, using the indicated antibodies. (B) The …
Figure 3—figure supplement 2
Enzymatic activity of mutant fumarases.
B. subtilis wild type (WT), Δfum, H127R and H186N strains were grown to logarithmic phase harvested and resuspended in PBS. The cells were lysed and centrifuged to obtain the supernatant which was …
Figure 3—figure supplement 3
mutant fumarases.
B. subtilis wild type, Δfum and mutant H127R fumarase strains were grown to logarithmic phase. MMS was added to final concentration of 0.07% (v/v) for 45 min; the cells were washed and serially …
Figure 3—figure supplement 4
mutant fumarases.
Wild type and Δfum strains were grown to logarithmic phase, MMS was added to final concentration of 0.07% (v/v) for 45 min. The cells were washed and serially diluted onto LB plates containing 250 …
Figure 4 with 1 supplement
Absence of fumarase affects RecN localization.
(A) Fluorescence microscopy images of B. subtilis expressing RecN-GFP in the absence (control) and presence of MMS (upper two rows of panels) and strains lacking the fumarase gene (Δfum, lower two …
Figure 4—figure supplement 1
- Absence of fumarase affects the number of RecN foci.
The proportion of B. subtilis cells showing RecN-GFP foci with or without DNA damage induction by MMS (the RecN-GFP in WT cells shows a proportion of 0.37–0.46 which is normalized to one). Error …
Figure 5
Fumarase affects RecN levels.
(A) RecN level is increased upon DNA damage. Wild type and Δfum strains, expressing RecN-GFP, were grown to logarithmic phase and MMS was added to final concentration of 0.07% (v.v). The cells were …
Author response image 1
Additional files
-
Supplementary file 1
B. subtilis strains.
B. subtilis strains used in this study and their source.
- https://doi.org/10.7554/eLife.30927.018
-
Supplementary file 2
S. cerevisiae strains.
S. cerevisiae strains used in this study and their source.
- https://doi.org/10.7554/eLife.30927.019
-
Transparent reporting form
- https://doi.org/10.7554/eLife.30927.020
