Significance Ribonucleotide reductases (RNR) play a critical role in supplying cellular deoxynucleotide pools. Nucleotide reduction by class Ia RNR requires a diferric-tyrosyl radical cofactor, which is a target of anticancer agents. How this essential cofactor is assembled in vivo is not well understood. We show here that a conserved protein complex composed of the Fe-S–requiring Dre2 and the diflavin-requiring Tah18, previously shown to donate electrons for Fe-S cluster assembly for proteins found in the cytosol and nucleus, also is required for RNR cofactor assembly. Deficiency in this complex leads to activation of both the DNA-damage checkpoint and the iron regulon, linking iron homeostasis to maintenance of genome stability. These findings may provide new insights into development of RNR-targeted therapeutics.