The DNA of Bacillus subtilis bacteriophage SP10 is partially resistant to cleavage and methylation in vitro by restriction enzyme R * BsuRI and its cognate methylase even though >20 copies of the target sequence, 5'.. . GGCC. .. 3', are present on the phage genome. YThy, a hypermodified oxopyrimidine that replaces a fraction of the thymine residues in SP10 DNA, was responsible for this protection, since YThy-free DNA was no longer resistant. Sites that were normally resistant could nevertheless be cleaved or methylated in vitro if the salt concentration was reduced or dimethyl sulfoxide was added to the reaction buffer. Analysis of the termini produced by cleavage suggested that resistant sites occurred in the sequence 5' .o.. GGCC-YThy ... 3', whereas sensitive sites, of which there were only two per genome, occurred in the sequence 5'. .. GGCCG. .. 3'. These in vitro results provide an explanation for the in vivo resistance of SP10 to restriction-modification by B. subtilis R. They also suggest ways in which the presence of the atypical base YThy in regions that flank the target might upset critical DNA-enzyme interactions necessary to locate and recognize the specific site of cleavage or methylation. YThy also strongly protected 5'. .. GCNGC. .. 3' (R * Fnu4HI) sequences on SP10 DNA, but the biological relevance of this protection is unclear.

Bacillus subtilis phage SP10 DNA has two oxopyrimidines, thymidine 5'-monophosphate (dTMP) and its hypermodified analog (YdTMP). Published data suggest that both are synthesized by postreplicational modification of 5-hydroxymethyldeoxyuridylate (HOMedUMP) in nascent DNA by the following pathway: HOMedUMP-*PPOMedUMP-*dTMP (85%) or YdTMP (15%); PPOMedUMP is 5-(hydroxymethyl-Opyrophosphoryl)deoxyuridylate, the pyrophosphoric acid ester of the C5CH2OH function of HOMedUMP. This paper describes aberrant DNAs synthesized at nonpermissive temperatures by a complementary series of heat-sensitive, modification-defective (mod) mutants. Collectively, these mutants encompass the major steps in the complete modification of nascent SP10 DNA. DNA produced by modA phage retains HOMedUMP as its sole oxopyrimidine, implying that (i) this mutant is defective in the pyrophosphorylation step and (ii) formation of PPOMedUMP is required for any further modification. Furthermore, studies with double mutants indicated that modA is epistatic for all other mod mutants, which supports the hypothesis that modA controls the earliest step in the modification pathway. Since their DNAs contain no YdTMP, modC and modD are defective in hypermodification (i.e., PPOMedUMP-YdTMP). However, dTMP occupies the entire oxopyrimidine fraction of modC DNA, whereas modD DNA has a normal dTMP content, but the now-missing YdTMP is replaced by either PPOMedUMP or a byproduct of abortive hypermodification. It is proposed that the modD mutants are defective in the catalytic aspects of hypermodification and that modC are defective in some regulatory function that promotes hypermodification at the expense of reductive modification (i.e., PPOMedUMP-*dTMP). Reductive modification is defective in modB phage, as evidenced by the absence of dTMP. In contrast to the others, modB DNA has a complex oxopyrimidine content: HOMedUMP, ca. 30%; PPOMedUMP, ca. 40%; and YdTMP, ca. 30%. The expanded level of YdTMP suggests that at certain sites, reductive modification and hypermodification are competing reactions. Interestingly, the PPOMedUMP content of modB DNA seemingly reflects the maximum degree to which phage DNA can be pyrophosphorylated, since the loss of YdTMP from modBmodC and modBmodD DNAs results in a unilateral increase in HOMedUMP content.