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

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.

Fumarase: From the TCA Cycle to DNA Damage Response and Tumor Suppression

“…Leshets et al, have found that yeast cytosolic fumarase is important for the HR repair pathway, through its function in the initial step of the DSB resection process (Figure 3 , right) (Leshets et al, 2018 ). Supporting this notion is that no genetic interactions were detected with the extensive resection factors Exo1 and Sgs1 (Leshets et al, 2018 ).…”

“…Leshets et al, have found that yeast cytosolic fumarase is important for the HR repair pathway, through its function in the initial step of the DSB resection process (Figure 3 , right) (Leshets et al, 2018 ). Supporting this notion is that no genetic interactions were detected with the extensive resection factors Exo1 and Sgs1 (Leshets et al, 2018 ). Moreover, previous publications indicated that during the initial step of resection 50 to 1,600 bases of DSB flanking DNA can be processed (Mimitou and Symington, 2008 ; Zhu et al, 2008 ; Garcia et al, 2011 ).…”

“…If following the depletion of cytosolic fumarase only the extensive step of resection is affected, one would detect at least some level of initial resection 0.29 kbp from the DSB. In fact, resection was not detected, suggesting that cytosolic fumarase is involved in the initial step of the DSB resection process (Leshets et al, 2018 ).…”

“…One hint was the reduced protein levels of Sae2 in cytosolic fumarase depleted cells, suggesting that fumarase acts upstream of Sae2, which we presume is regulated by determining its protein abundance. In this regard, cytosolic fumarase regulation of Sae2 is at the protein level, and not at the Sae2 mRNA level (Leshets et al, 2018 ). It is possible that cytosolic fumarase may enhance the translation of Sae2 or have a negative effect on its degradation.…”

The Landscape of Mutations in Human Fumarate Hydratase

“…As a result the enzyme FH has been described as a tumor suppressor, and fumarate as part of a novel classification of molecules named “oncometabolites”. Precisely how the buildup of fumarate can be oncogenic is unknown, but recent work points towards suppression of DNA repair responses, EMT, and promotion of mitotic entry upon fumarate buildup 3–5 .…”