Synthesis of deoxythymidylate and the unusual deoxynucleotide in mature DNA of Bacillus subtilis bacteriophage SP10 occurs by postreplicational modification of 5-hydroxymethyldeoxyuridylate.

Mature DNA of Bacillus subtilis W23 phage SP10 contains a hypermodified nucleotide (YdTMP) that replaces ca. 20% of the DTMP. SP10 DNA was pulse-labeled for 1 min at 20 degrees C with 32Pi. Among the oxopyrimidine nucleotides, virtually all of the radioactivity was recovered as 5-hydroxymethyldeoxyuridylate (HMdUMP). During the subsequent chase, radioactivity was lost from HMdUMP and recovered as YdTMP. At 37 degrees C, exogenous [6-3H]5-hydroxymethyldeoxyuridine (HMdUrd) was incorporated into SP10 DNA. Label administered as HMdUrd was phosphorylated to HMdUTP in the infected cells, but all radioactivity was recovered from SP10 DNA as YdTMP and dTMP. Two heat-sensitive mutants defective in hypermodification of SP10 DNA are described. In one mutant, HMdUMP replaces YdTMP in DNA. The other mutant generates a DNA containing a novel deoxynucleotide in place of YdTMP. The novel deoxynucleotide seems to consist of PPi esterified to the 5-hydroxymethyl function of HMdUMP (PP-HMdUMP). Both mutants make normal amounts of dTMP. The data are discussed in terms of the following conclusions. (i) Both oxopyrimidine nucleotides in mature SP10 DNA are derived by postreplicative modification of HMdUMP in nascent DNA. (ii) PP-HMdUMP is an intermediate that facilitate formation of a putative exocyclic methylene intermediate which receives the hypermodification. It is also argued that PP-HMdUMP and the same exocyclic methylene intermediate could serve as intermediates in reductive modification to dTMP. (iii) YdTMP is not an intermediate in the formation of dTMP, and reductive modification proceeds independently of hypermodification.