Abstract
TagI belongs to the recently characterized SRA-HNH family of modification-dependent restriction endonucleases (REases) that also includes ScoA3IV (Sco5333) and TbiR51I (Tbis1). Here, we present a crystal structure of dimeric TagI, which exhibits a DNA binding site formed jointly by the nuclease domains, and separate binding sites for modified DNA bases in the two protomers. The nuclease domains have characteristic features of HNH/ββα-Me REases, and catalyze nicks or double strand breaks, with preference for /RY and RYN/RY sites, respectively. The SRA domains have the canonical fold. Their pockets for the flipped bases are spacious enough to accommodate 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC), but not glucosyl-5-hydroxymethylcytosine (g5hmC). Such preference is in agreement with the biochemical determination of the TagI modification dependence and the results of phage restriction assays. The ability of TagI to digest plasmids methylated by Dcm (C5mCWGG), M.Fnu4HI (G5mCNGC) or M.HpyCH4IV (A5mCGT) suggests that the SRA domains of the enzyme are tolerant to different sequence contexts of the modified base.
Highlights
Modification of DNA is commonly occurring in phages, and examples have been found for variants of all four standard DNA bases in their genomes
TagI was recombinantly overexpressed from a codon-optimized synthetic gene in E. coli, as an inteinbased self-cleaving chitin binding domain fusion protein
All assays with the purified protein were carried out at 37◦C
Summary
Modification of DNA is commonly occurring in phages, and examples have been found for variants of all four standard DNA bases in their genomes. Phages or other mobile elements can acquire DNA modifications by passage in the host, or by incorporation of modified dNTPs into DNA, which can be optionally modified further. Modified bases in phage or other mobile DNA provide protection against ‘conventional’ host restriction. 2 -deoxyguanosine replacement by 2 -deoxyarchaeosine (dG+) in the Escherichia coli phage 9g DNA renders it resistant to over 70% of commercially available Type II restriction endonucleases (REases) [7]. In addition to protection against conventional restriction systems, modified DNA bases may have other functions, for example may facilitate packing of DNA in the phage head [9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
