Wiatr C L, Witmer H J
J Virol. 1984 Oct;52(1):47-54. doi: 10.1128/JVI.52.1.47-54.1984.
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 greater than 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' ... 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.
枯草芽孢杆菌噬菌体SP10的DNA在体外对限制性内切酶R. BsuRI及其同源甲基化酶的切割和甲基化具有部分抗性，尽管噬菌体基因组上存在超过20个靶序列5'... GGCC... 3'的拷贝。YThy是一种高度修饰的氧嘧啶，它取代了SP10 DNA中一部分胸腺嘧啶残基，导致了这种保护作用，因为不含YThy的DNA不再具有抗性。如果降低盐浓度或在反应缓冲液中加入二甲基亚砜，通常具有抗性的位点在体外仍可被切割或甲基化。对切割产生的末端进行分析表明，抗性位点出现在序列5'... GGCC - YThy... 3'中，而敏感位点（每个基因组仅有两个）出现在序列5'... GGCCG... 3'中。这些体外实验结果解释了SP10在体内对枯草芽孢杆菌R的限制修饰作用具有抗性的原因。它们还表明，靶序列侧翼区域中存在的非典型碱基YThy可能会破坏定位和识别切割或甲基化特定位点所需的关键DNA - 酶相互作用。YThy也强烈保护SP10 DNA上的5'... GCNGC... 3'（R. Fnu4HI）序列，但这种保护作用的生物学意义尚不清楚。
