Editors' ChoiceDNA damage

A metabolite rescues DNA damage

Sci. Signal.  15 Sep 2015:
Vol. 8, Issue 394, pp. ec264
DOI: 10.1126/scisignal.aad3684

DNA double-strand breaks (DSBs) can be repaired through nonhomologous end-joining (NHEJ), during which a heterodimer of Ku70 and Ku80 is recruited to DSB regions, which in turn recruit other NEHJ factors, including the catalytic subunit of DNA-dependent protein kinase (DNA-PK). Fumarase (FH) is a mitochondrial enzyme that reversibly converts fumarate to malate, metabolites that participate in the generation of ATP by the tricarboxylic acid (TCA) cycle. Compared with wild-type yeast, yeast with a deletion of FUM1, which encodes FH, accumulates more DNA damage in response to ionizing radiation (IR). Jiang et al. (see also Lees-Miller) investigated a role for FH in DSB repair in mammalian cells. Immunoblotting analysis of chromatin extracts from cell-cycle synchronized; human osteosarcoma (U2OS) cells showed that IR enhanced the binding of FH to chromatin in G1 and other stages of the cell cycle. Analysis of Flag-tagged FH immunoprecipitates from U2OS cells showed that IR increased the association of FH with the histone variant H2A.Z, which induces reorganization of chromatin architecture at DSB regions to facilitate DNA repair. H2A.Z depletion by shRNA in U2OS cells reduced the recruitment of FH, Ku70, and Ku80 to DSB regions induced by exposure to IR. Cells exposed to the DNA-PK inhibitor NU7441 and then to IR showed reduced association of FH with chromatin, suggesting that DNA-PK activity was important for recruiting FH to DSB sites. In vitro kinase assays revealed that DNA-PK phosphorylated FH at Thr236. Preincubation of U2OS cells with NU7441 reduced IR-induced binding of FH to H2A.Z, and treating immunoprecipitated FH with calf alkaline phosphatase reduced the amount of associated H2A.Z. Furthermore, expression of a T236A mutant of FH, which could not be phosphorylated by DNA-PK, showed less coimmunoprecipitation of FH and H2A.Z from irradiated U2OS cells, compared with amount that coprecipitated from cells expressing wild-type FH. Cells expressing a catalytically inactive form of FH that bound to chromatin or the T236A mutant, which is catalytically active but had reduced H2A.Z association, exhibited less NHEJ, suggesting a role for nuclear fumarate in DSB repair. Exogenous application of fumarate into the cell medium resulted in Ku70 accumulation at sites of DNA damage and in partial rescue of NHEJ in irradiated U2OS cells reconstituted with catalytic inactive FH or the T236A mutant. H3K36 dimethylation (H3K36me2) increases at DSB regions, which promotes the association of Ku70 with these sites, and this response was reduced in irradiated U2OS cells expressing either a catalytically inactive FH mutant or the T236A FH mutant. As expected, ectopic expression of the demethylase that acts on H3K36me2 (KDM2B) in U2OS cells reduced H3K36me2 abundance at IR-induced DSB regions. This effect was partially reversed by exogenous addition of fumarate in a dose-dependent manner. These results indicated that the DNA-PK-mediated phosphorylation of FH promotes its recruitment to DSB regions, where it generates fumarate to promote NHEJ.

Y. Jiang, X. Qian, J. Shen, Y. Wang, X. Li, R. Liu, Y. Xia, Q. Chen, G. Peng, S.-Y. Lin, Z. Lu, Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation. Nat. Cell Biol. 17, 1158–1170 (2015). [PubMed]

S. P. Lees-Miller, Fumarate in DNA repair. Nat. Cell Biol. 17, 1096–1097 (2015). [PubMed]