Significance of ribonucleotide reduction in the biosynthesis of deoxyribonucleotides in Escherichia coli.

Abstract

A pyrimidine auxotroph of Escherichia coli, possessing decreased deaminase activities for cytidine and deoxycytidine, was grown with uniformly 14C‐labeled cytidine or uridine as sole pyrimidine source. The incorporation of radioactivity into the individual mononucleotides of DNA and RNA was determined. The pyrimidine nucleotides were degraded, and the distribution of isotope between their pentose and base moieties was analyzed.[14C]‐Cytidine was incorporated into RNA without loss of its isotope content. Furthermore, CMP of RNA and deoxyCMP of DNA had identical specific activities and pentose: base ratios. Therefore deoxyCMP of DNA was formed exclusively by reduction of a cytidine derivative.Only approximately 20% of the pentose isotope content of [14C]‐uridine was retained after incorporation into RNA. CMP, UMP, deoxyCMP, and deoxyTMP isolated from the nucleic acids had approximately equal specific activities and pentose: base ratios, indicating that both pyrimidine deoxyribonucleotides were formed by ribonucleotide reduction.[14C]‐Cytidine was converted to UMP of RNA with almost complete loss of isotope from the ribosyl moiety, but was converted to deoxyTMP of DNA with little loss of label. After estimation of different enzymatic activities in cell‐free extracts it was concluded that the major pathway yielding deoxyTMP of DNA did not involve deoxyTMP synthetase but rather proceeded directly from a deoxycytidine compound.