Selective protection of 5' ... GGCC ... 3' and 5' ... GCNGC ... 3' sequences by the hypermodified oxopyrimidine in Bacillus subtilis bacteriophage SP10 DNA

Abstract

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.

Restriction and modification of SP10 phage byBsuM ofBacillus subtilisMarburg

“…SP10 phage genome DNA (G + C, 42 mol%) contains incompletely characterized hypermodified thymine (YThy) residues in an amount of 5% in addition to 21% thymine residues [18]. The hypermodification Y is composed of L ‐glutamate and a basic component, and hypermodification‐defective mutant SP10 phages are isolated [19].…”

“…So some BsuM recognition sites of SP10 phage genome may not be methylated but protected in the BSU59 (Dter(groESL)) strain by BsuMM protein from BsuMR digestion. SP10 phage genome DNA (G + C, 42 mol%) contains incompletely characterized hypermodified thymine (YThy) residues in an amount of 5% in addition to 21% thymine residues [18]. The hypermodification Y is composed of L-glutamate and a basic component, and hypermodification-defective mutant SP10 phages are isolated [19].…”

α-Putrescinylthymine and the sensitivity of bacteriophageφW-14 DNA to restriction endonucleases

“…Type II restriction endonucleases are affected by the sequences flanking their recognition sites (4-8, 17-24), by DNA conformation (25)(26)(27), and by base modification (2)(3)(4)(5)(6)17,20,24,(28)(29)(30)(31). EcoRI is, perhaps, the best understood of these enzymes.…”

“…As might be expected, since methylation of specific cytosines (C) or adenines (A) within the palindromic recognition sequences of Type II restriction endonucleases will prevent cleavage by certain of the enzymes, the modified phage DNAs of both types are either resistant to, or are cleaved only slowly by, many of these enzymes (2-6). Since sequences lying outside the recognition site influence the cleavage of DNA by EcoRI (7,8), it is not surprising that the modified bases can, in at least some instances, protect sites which do not contain the base in question (4)(5)(6).…”

“…The modifying groups in those DNAs examined vary considerably in size, and the majority of them are uncharged (1)(2)(3)(4)(5)(6). The DNA of Bacillus subtilis phage SP15 is unusual in that 62% of its thymine (T) residues are replaced by phosphoglucuronated and glucosylated 5-(4',5'-dihydroxypentyl)uracil (hp U; 9,10).…”