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

Journal Article · 14 April 2025 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkaf291 · OSTI ID:2556908

Shen, Betty W.; Heiter, Dan; Yang, Weiwei; Xu, Shuang-yong; Stoddard, Barry L.

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.

• Sponsoring Organization: USDOE
• OSTI ID: 2556908
• Journal Information: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 7 Vol. 53; ISSN 0305-1048
• Publisher: Oxford University PressCopyright Statement
• Country of Publication: United Kingdom
• Language: English