Abstract
To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5-NedU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among >200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC (TS, S=C, or G), implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine.
Highlights
Type II restriction and modification (R-M) systems in bacteria encode restriction endonucleases (REases) to destroy invading foreign DNA in phage infection and acquisition of mobile genetic elements (Smith and Wilcox, 1970; Landy et al, 1974, reviewed in Pingoud et al, 2016)
To find out the resistance level, we carried out restriction digestions for phage M6, ViI, and phi W-14 genomic DNA
Phi W-14 DNA is resistant to restriction by Hpy188III (TCNNGA) and HpyCH4V (TGCA) probably due to the modified bases in TG or TC dinucleotides in both strands
Summary
Type II restriction and modification (R-M) systems in bacteria encode restriction endonucleases (REases) to destroy invading foreign DNA in phage infection and acquisition of mobile genetic elements (Smith and Wilcox, 1970; Landy et al, 1974, reviewed in Pingoud et al, 2016). Phage T4 DNA modified with 5gmC is resistant to many Type II REases that recognize GC-containing sequences (Huang et al, 1982). In a limited Type II restriction study of phage phi W-14 genome containing putT, 17 out of 32 REases tested were blocked by the base modification (Miller et al, 1985). Phage SPO1 genomic DNA with 5hmdU substitutions is fully resistant to 4 out of 30 Type II restrictions (∼13.3%) and partially resistant (i.e., slower cleavage in 1 h digestion) to 17 out of 30 REases tested (56.7%) (Huang et al, 1982; Vilpo and Vilpo, 1995). DNA duplex oligos with the 5hmdU substitution display reduced melting temperature (Tm) and altered backbone flexibility when passing through nanopores (Carson et al, 2016)
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