Fe-S cluster biogenesis machinery is required for multiple DNA metabolism processes. In this work, we show that, in Saccharomyces cerevisiae, defects at different stages of the mitochondrial Fe-S cluster assembly machinery (ISC) result in increased spontaneous mutation rate and hyper-recombination, accompanied by an increment in Rad52-associated DNA repair foci and a higher phosphorylated state of γH2A histone, altogether supporting the presence of constitutive DNA lesions. Furthermore, ISC assembly machinery deficiency elicits a DNA damage response that upregulates ribonucleotide reductase activity by promoting the reduction of Sml1 levels and the cytosolic redistribution of Rnr2 and Rnr4 enzyme subunits. Depending on the impaired stage of the ISC machinery, different signaling pathway mediators contribute to such a response, converging on Dun1. Thus, cells lacking the glutaredoxin Grx5, which are compromised at the core ISC system, show Mec1- and Rad53-independent Dun1 activation, whereas both Mec1 and Chk1 are required when the non-core ISC member Iba57 is absent. Grx5-null cells exhibit a strong dependence on the error-free postreplication repair and the homologous recombination pathways, demonstrating that a DNA damage response needs to be activated upon ISC impairment to preserve cell viability.