A protein architecture guided screen for modification dependent restriction endonucleases

Abstract

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines

“…endonucleases (Lutz et al, 2019). Many more Type IV restriction systems have been found to restrict 5mC and 5hmC modified DNA (Loenen and Raleigh, 2014).…”

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

“…Unmodified DNA or PT backbone modification in a different sequence context would serve as "self ". For example, Streptomyces coelicolor A3 contains only 4mC modified bases and no PT modifications in its genomic DNA (dnd − ) (Lutz et al, 2019). ScoMcrA endonuclease encoded by its genome restricts dnd + plasmid (GpsGCC/GpsGCC) coming from Streptomyces lividans that contains dndA-E modification genes (He et al, 2007).…”

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA

“…A role of the enzymes as MDREs is consistent with the spotty phylogenetic distribution and a lack of accompanying DNA MTases. It is also supported by lack of toxicity to unprotected hosts, (moderate) modification dependent DNA endonuclease activity in vitro , and (moderate) modification dependent restriction activity in vivo ( 18 , 19 ).…”

“…The superfamily comprises several other domain families, including YTH and EVE domains, that are so far predominantly associated with binding of modified RNA nucleobases ( 15 , 16 ), despite hints that point to a role in DNA biology ( 17 , 18 ). Recently, it has become clear that in many eu- and archaebacteria, PUA superfamily domains cooperate with PD-(D/E)XK or HNH nuclease domains, either as fused proteins ( 19 ) or in dedicated protein complexes ( 18 ). The fusion proteins could be directed against RNA or DNA.…”

“…In high concentration the enzyme cleaves any DNA, but dilution series demonstrate a preference for 5hmC over g5hmC containing or unmodified DNA ( 19 ). Consistently, TspA15I expression in E. coli cells protected against T4gt phage (which due to glucosyltransferase deficiency contains 5hmC), with lower efficiency against T4 phage (which contains g5hmC), but not against λvir phage ( 19 ). Structures that could shed light on the mode of binding of the EVE domain to DNA containing modified cytosine bases are not available.…”