# Cryo-EM structures of DNA-free and DNA-bound BsaXI: architecture of a Type IIB restriction–modification enzyme

## TL;DR

This study reveals the structure of the BsaXI enzyme with and without DNA, showing how it changes shape to cut DNA at specific sites.

## Contribution

The paper presents novel cryo-EM structures of BsaXI and identifies key residues involved in DNA cleavage.

## Key findings

- BsaXI forms a stable heterotrimeric complex with or without DNA.
- DNA binding induces a conformational shift from open to closed states.
- Two residue clusters in the RM subunit are critical for DNA cleavage.

## Abstract

We have determined multiple cryogenic electron microscopy (cryo-EM) structures of the Type IIB restriction–modification enzyme BsaXI. Such enzymes cleave DNA on both sides of their recognition sequence and share features of Types I, II, and III restriction systems. BsaXI forms a heterotrimeric (RM)2S assemblage in the presence and absence of bound DNA. Two unique structural motifs—a multi-helical “knob” and a long antiparallel double-helical “paddle”—are involved in DNA binding and cleavage. Binding of the DNA target triggers a large conformational change from an ‘open’ to ‘closed’ configuration, resulting in a mixture of two different conformations with respect to the positioning of the S subunit and its target recognition domains on the enzyme’s bipartite DNA target site. Structure-guided mutagenesis studies implicated two clusters of residues in the RM subunit as being critical for DNA cleavage, both are located proximal to a DNA cleavage site. One corresponds to a canonical PD-(D/E)xK endonuclease site in the N-terminal endonuclease domain, while the other corresponds to residues clustered within the paddle motif (near to the C-terminal end of the RM subunit). This analysis facilitates a comparison of three potential mechanisms by which such enzymes cleave DNA on each side of the bound target.

Graphical Abstract