Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R

Abstract

Hi, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B. subtilis R cells. In vitro, Hi DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of-GGCC-cleavage sites was approximately 250. However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol. After the substitution of thymine for HMU by DNA cloning in B. subtilis, a BsuR cleavage site was restricted and modified under standard conditions. No additional sites were detected after shotguncloning of about 11% of the chromosome. The nucleotide sequence of a cleavage site was found to be 5'. .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-CC -Hmu-A-G-...3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences. The results suggested that the resistance of Hi to restriction and modification by B. subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites.

Nichtkanonische Basen im Genom: die regulative Informationsebene in der DNA

“…In Phagen wird dabei zunächst das monomere dT‐Nukleotid zu hmdU oxidiert und dann als das jeweilige Triphosphat in das Genom eingebaut . Die Base schützt das Phagengenom vor Abbau durch Restriktionsenzyme des Wirts . Zusätzlich kann man hmdU und seine glukosylierte Version, besser bekannt als Base J, im Genom von Kinetoplastiden finden .…”

Specificity of hydroxylmethyluracil-containing DNA for transcription factor 1: Structural insights

“…Hydroxymethyluracil‐containing DNA has been shown to be a good template‐primer for various DNA and RNA polymerases,12 and serves as a preferred‐binding site (over its thymine‐DNA counterpart) for phage‐encoded proteins13, 14 while disrupting other protein‐DNA interactions 15. Indeed, transcription factor 1 (TF1), an SPO1 bacteriophage‐encoded protein, has been shown to bind cooperatively and specifically, only to hmU‐DNA but not T‐DNA 16.…”

Cloning of the resistant EcoRII recognition site of phage T7 into an EcoRII‐sensitive plasmid makes the site susceptible to the restriction enzyme

“…T7 DNA is sensitive to BstNI but resistant to EcoRII despite not being Dcm-methylated (KRUGER et al 1985).This raised the general question of why certain DNA sequences which are neither methylated nor contain unusual bases (for a review about these protection mechanisms see KRUGER and BICKLE 1983) are nevertheless refractory to a restriction endonuclease. The literature states examples where sequences flanking the recognition site exert an influence on their susceptibility to cleavage by a certain restriction enzyme ( BRON et al 1983, BUSSLINGER et al 1983, GINGERAS and BROOKS 1983, KESHET and CEDAR 1983.We cloned a 219 bp fragment of T7/Bln DNA (from our T7/BERLIN strain) with the EcoRII site in its center into the vectors mp18 and pUC18. Fig.…”