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

Abstract

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.

A Unique m6A-Dependent Restriction Endonuclease from an Archaeal Virus

“…All of these SRA domain-containing endonucleases cleave DNA at a certain distance away from the modified cytosine ( 10 ). In addition, SRA domains belong to the PUA superfamily that includes other modified DNA/RNA-binding domains, such as EVE domains and YTH domains ( 2 , 12 , 13 ). These domains can also be fused to endonucleases, and most of the fusion proteins function as cytosine-modification-specific endonucleases ( 2 , 12 ).…”

“…In addition, SRA domains belong to the PUA superfamily that includes other modified DNA/RNA-binding domains, such as EVE domains and YTH domains ( 2 , 12 , 13 ). These domains can also be fused to endonucleases, and most of the fusion proteins function as cytosine-modification-specific endonucleases ( 2 , 12 ). Compared to the characterized cytosine modification-dependent endonucleases, very few m6A-dependent restriction endonucleases have been identified, and DpnI is the best-known example ( 2 , 14 to 17 ).…”

Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems

“…Since the PCR products contain 5mC and 5hmC in many different contexts, this result suggests that the enzyme has no activity against methylated or hydroxymethylated DNA, despite the presence of the PUA (SRA-like) domain. This was surprising because it had been shown previously that PUA superfamily REases VcaM4I, SRA-like domain-containing endonuclease TagI, and PvuRts1I restricted DNA containing modified cytosines (Janosi et al, 1994;Pastor et al, 2021). Possible activity against WT T4 [glucosylated(g)-5hmC] modified DNAs remains to be tested.…”

Prokaryotic winged helix domains as dsDNA adenine methylation sensors

“…How the modification sensor domain keeps the activity of the nuclease domain in check is not well understood. However, in some cases it can be shown that the linker has an inhibitory role for the endonuclease that is only relieved once modified DNA is bound to the reader domain and the complex reorganizes structurally (19). The PUA-wH-HNH (phi.HhiV4I) and wH-GIY-YIG (Ahi29725I and Apa233I) that were tested by run-off sequencing are consistent with this expectation.…”

“…PUA superfamily sensors comprise SRA domains with specificity for 5mC, as in MspJI (14), and related domains (15,16), originally also termed SRA domains, with specificity for 5-hydroxymethylcytosine (5hmC) and glucosyl-5-hydroxymethylcytosine (g5hmC), as in the PvuRts1I family of restriction endonucleases (17,18). The PUA superfamily also comprises EVE domains specific for 5mC and 5hmC, as found in VcaM4I (19), and YTH domains (Yth-McrB fusion) specific for 6-methyladenine (6mA) (20)(21)(22), as well as ASCH domains. Bioinformatic analysis has suggested that ASCH domains might be 6mA readers (23), but this prediction is not confirmed by experimental data so far.…”