Perturbation of the deoxyribonucleotide triphosphate (dNTP) pool is recognized for contributing to the mutagenic processes involved in oncogenesis. The RAS gene family encodes well-characterized oncoproteins whose structure and function are among the most frequently altered in several cancers. In this work, we show that fluctuation of the dNTP pool induces CG → TA mutations across the whole genome, including RAS gene at codons for glycine 12 and 13, known hotspots in cancers. Cell culture addition of the ribonucleotide reductase inhibitor thymidine increases the mutation frequency in nuclear DNA and leads to disruption of mitochondrial metabolism. Interestingly, this effect is counteracted by the addition of deoxycytidine. Finally, screening for the loss of hydrogen bonds detecting CG → TA transition in RAS gene of 135 patients with colorectal cancer confirmed the clinical relevance of this process. All together, these data demonstrate that fluctuation of intracellular dNTP pool alters the nuclear DNA and mitochondrial metabolism.