restriction enzyme bsaxi  (New England Biolabs)

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Cryo-EM data collection, refinement, and validation statistics of BsaXI

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques:

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Structure of the DNA-free BsaXI enzyme assemblage. ( A ) Density map of DNA-free BsaXI at 3.2 Å resolution in two different orientations. ( B ) Local resolution of the density map ranging from better than 3 Å at the core to 6 Å at the periphery. ( C ) Segger segmentation map of apo-BsaXI in two different orientations, showing four contiguous sections corresponding to the two RM subunits (cyan and light purple) and the tip of the two TRD domains of the S subunit. ( D ) Overlay of the final apo-BsaXI model with the density map. The REase domains and the tip of the paddle domains are shown outside of the density at 7 sigma level. ( E ) Final ribbon model of the apo-BsaXI in two different orientations. In the corresponding PDB file, the RM subunits contacting TRD1 and TRD2 are assigned as chain A (purple) and chain B (orange), and the S subunit (green) is assigned as chain C. ( F ) Ribbon model of the S subunit showing TRD1-CR1 (green) and TRD2-CR2 (purple). ( G ) Density maps corresponding to the S subunit and its two TRD-CR domains.

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques:

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Details of the RM subunit of DNA-free BsaXI. ( A ) Ribbon diagram of RM subunit. Endonuclease: light gray; multiple-helix-connector domain: cyan; methyltransferase: purple; knob: forest green; loops: earth; and paddle: blue. Inset: Schematic representation of the BsaXI model. ( B ) Representative maps of different regions of the DNA-free enzyme. ( C ) Topographical schematic of the N 6 -adenosine methyltransferase domain with the central eight strand β-sheet in green and the five flanking helices in blue, solid outlined helices in the front, and dash-outlined ones in the back. The knob domain is inserted between the 7th and the 8th β-strands while the antiparallel double-helix paddle extends from the end of the 8th strand.

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques:

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Intersubunit interactions and conformational changes in S subunit upon DNA binding. (A) Interactions between the RM subunits and the TRDs of the S subunit in the DNA-free BsaXI, showing the intercalation of the TRD domains into the hydrophobic pocket at the boundary of the MTase, loop, and knob domains. Top panels : The RM subunits are colored according to domains: MTase: dark purple; knob domain: light purple, loops: orange; paddle: blue; S subunit: green. Bottom panels : The surface of the RM subunits is colored according to hydrophobicity of the surface residues. ( B, C ) Superposition of the DNA-free BsaXI S subunit (purple) with the S subunit of the DNA-bound (green) showing different rotation of the TBD domains in the two conformers upon binding to the bipartite DNA target. ( B ) Superposition of S subunits from DNA-free BsaXI and DNA-bound conformer I show close alignment of TBD1 domain and CCW rotation of the TBD2 domain (purple curved arrow). ( C ) Superposition of S subunits from DNA-free BsaXI to DNA-bound conformer II show close alignment of TBD2 and CCW rotation of TBD1.

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques: Binding Assay

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Cryo-EM structures of DNA-bound BsaXI. ( A ) Two different orientations of the initial 3D volume for BsaXI–DNA particle, showing the “Closed” conformation, the long handlebar configuration of the S-subunit, and the supercoil double helix of the DNA. ( B ) Left : Cryo-EM map of DNA-free BsaXI overlaid with ribbon representation the (RM) 2 S protein structure. Right : Map if the BsaXI/DNA complex (overlaid with the ribbon representation of the initial model for the DNA-bound conformation of the (RM) 2 S-DNA complex. ( C ) 3D variability analysis of the DNA-bound enzyme complex, showing the presence of two populations of particles before and after heterogeneous refinement (top and bottom, respectively). ( D ) Superposition of the maps corresponding to the S-subunits in the two DNA-bound conformation after heterogenous refinement (blue and green), versus the initial map (gray), showing different disposition of the TRDs. ( E ) Comparison of the refined maps of the two different DNA-bound enzyme conformations. Orange and blue circles highlight the presence and absence of density for the REase domain. ( F ) Schematic presentation of conversion from DNA-free to DNA-bound BsaXI particles deduced from cryo-EM maps. The endonuclease domain (‘R’) visible at one of two positions in each DNA-bound conformation is indicated by a highlighted circle at each position. ( G ) Domain organization of BsaXI–DNA complex with the S-subunit removed from the top layer, showing a multiple-colored ribbon model of RM* subunit A, a ribbon model of RM subunit B in gray, and the double-stranded DNA. The top DNA strand is beige, and the compliment strand is orange. The upstream AC target is on the left and CTCC target on the right. The RM* subunit that interacts with the flipped-out bases is depicted in multicolor ribbon. The nuclease domain REase* is pea green, followed by the multiple helical connector* (cyan), MTase* (purple), the knob* (forest green), and the Paddle (blue). The long antiparallel double helix Paddle domain (corn flower blue) is tethered to the knob region by a network of loops (yellow). The upstream AC target is on the left with the flipped-out adenine base (space filling) inserts into the MTase* domain. The N 6 -methyl-adenine nucleotide (space filling) in the complement to the downstream (CTCC) target is on the right pointing toward the multi-helical connector* (HC*; cyan ribbon). The other RM subunit (light gray) is antiparallel to chain A with the MTase (M ) domain (purple outline) directly above the multi-helix connecter* (cyan ribbon); the multi-helix connecter (cyan outline) of the other subunit is positioned against the MTase* domain of the RM* subunit. ( H ) Close-up views of the two adenine bases [one in the 5′ AC target region ( upper panel ) and the second in the opposing strands of the 3′ CTCC target region ( lower panel )] that are each flipped out of the DNA duplex and located in binding pockets formed by one or both RM subunits, and targeted for methylation. The former base is methylated, while the second is unmethylated; they are located proximal to bound S-adenosyl-L-homocysteine (SAH) and SAM cofactor molecules that are observed in each of the two respective MTase active sites.

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques: Cryo-EM Sample Prep, Comparison, Binding Assay, Methylation

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Protein–DNA interactions in the two conformations of the DNA-bound BsaXI complex. ( A ) Direct contacts made by residues from chains A (RM subunit 1), B (RM subunit 2), and C (S subunit) are marked in black, blue, and green, respectively. The four magenta open arrows at the ends indicate the position of the scissile phosphodiester linkages. The red lines indicated nonspecific hydrophilic (electrostatic) interactions between the rotamer and/or peptide backbone of proteins and the DNA phosphate back bone. The numbering next to the phosphate backbone indicate the position of the base pairs relative to the flipped-out adenine or 6MA. For clarity, only some of the nonspecific electrostatic interactions are indicated. The green and pink filled circles highlighted the surrounding of the flipped-out adenine and the N 6 -methylated-adenine (6MA) in the two conformers. The S subunit utilizes identical residues in direct contacts with the DNA target in both bound conformations. Two residues (Y169 and R112) in the S subunit contact the upstream AC target site and five residues (R351, D376, T416, M417, and K419) contact the downstream CTCC target site. Almost all contacts between an RM subunit and DNA are exclusively made by one or the other subunit in the separate bound conformations. The flipped-out adenine is stabilized by F356, N523, P524, F526, and T612 from the MTase* domain. The flipped-out 6MA is surrounded and stabilized by four residues (R350, H221, N222, and W225) from the multi-helical connector domain. ( B, C ) Density features surrounding the flipped-out adenine (left) and N 6 -methylated-adenine (right).

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques: Methylation

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Mutational effect of alanine substitution at residues in the N-terminal endonuclease domain and C-terminal paddle domain. ( A ) Two views, from different perspectives, of the interaction of a portion of an RM subunit near one of the two DNA cleavage sites (upstream of the 5′ AC 3′ target region), showing the proximity of the N-terminal endonuclease domain (REase) and C-terminal paddle region to the two DNA strands near the cleavage site. ( B ) Residues in the REase domain and paddle region that were individually subjected to single-site alanine point mutagenesis. Those positions that resulted in the inactivation of DNA cleavage activity upon mutagenesis are indicated in red font. ( C ) Phage λ DNA (which contains 19 BsaXI target sites and runs as a single linear species near the top of the gel in the absence of enzyme) was digested with WT reconstituted BsaXI (leading to complete digestion and the appearance of multiple DNA fragments of reduced size and increased electrophoretic mobility; right side of lower gel) and with similar concentrations of a panel of individual alanine point mutants placed at individual positions in the endonuclease domain (D57A through E120A) and the paddle region (D811A through E823A). Mutations at four positions in the endonuclease domain (E73A, D90A, E105A, and K107A) and two positions in the paddle region (D816A and E823A) cause obvious, complete inactivation of the enzyme. Additional cleavage experiments using plasmid substrates (not shown) show no indication of nickase activity above background, indicating that DNA cleavage appears to be fully suppressed by these mutations. (Note that D816A mutant still has a low partial activity.)

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques: Mutagenesis, Activity Assay, Plasmid Preparation

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Hemimethylation on either target strand is sufficient to prevent DNA cleavage to either side of the enzyme’s target site. Four separate fluorescently labeled dsDNA duplexes (each containing a central bipartite target site for BsaXI, in each of four possible methylation states) were digested with BsaXI as described in the ‘Materials and methods’ section, and the resulting digest products were visualized and quantitated using CE analysis. While the unmethylated dsDNA substrate is cleaved to completion to either side of the target site, both hemi-methylated substrates and the fully bi-methylated substrates show no sign of cleavage in this experiment.

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques: Labeling, Methylation

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: Summary of AlphaFold structure predictions for identified Type IIB RM systems. ( A ) AlphaFold models of Type IIB enzyme homologues with Type I-like RM/S subunit compositions. Left panel : Overlay of predicted models for RM fusion of BcgI (badge), BveI (cyan), Hso63250II (pink) and HpyUM (green) showing the excellent superposition of all R (REase), H (multi-helical connector; MHC), and M (MTase) domains except the REase of Bcg I and the very flexible C-terminal tails. Right panel top : Predicted model for the S subunit of BveI showing the TRDs and antiparallel CR1 and CR2. Right panel bottom : predicted model for the S subunit of BcgI with skewed CR1 and CR2. The distorted arrangements of the two TBDs and CRs in the skewed S subunit could be the reason why BcgI requires higher order arrangements for activity. ( B ) Overlay of examples of predicted models of Type IIB RM system with a single-chain RMS composition, indicating the presence of the knob domain (orange circle) and the characteristic (TRD1–CR1–TRD2–CR2) fold of the Type I HsdS subunit. The latter is tethered to the MTase domain via a flexible loop. ( C ) Superposition of the predicted model and the cryo-EM structure of BsaXI (light and dark blue, respectively). All the predicted models of the identified Type IIB with single chain chimeric RMS configuration have a knob domain (orange circle), but BsaXI is the only Type IIB with both the knob (orange circle) and the long double helix-paddle (cyan circle) domains as observed in the cryo-EM structure. ( D ) Predicted structure of RdeGBIII, the Type IIB RMS fusion peptide with the S domain resembles that of the Type IIG proteins BpuSI and MmeI. ( E ) X-ray crystal structure of BpuSI.

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques: Activity Assay, Cryo-EM Sample Prep

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf291

Figure Lengend Snippet: PROMOLS3D based phylogenetic tree of 94 protein sequences that share >50% identity to BsaXI. The orange and blue color wedges indicate protein sequences with < or >800 residues, respectively. The filled orange and blue wedges indicated sequences randomly selected for AlphaFold structure prediction. Results from structure predictions showed that the orange-colored shorter sequences contain, in addition to the highly conserved R, H, and M domains, a knob domain. All shorter entries, with only four exceptions, segregated to a third of the phylogenetic tree circle. The longer protein sequences including BsaXI (in red letters) uniformly contain both the knob and paddle folds, and are phylogenetically further away from the shorter orange species.

Article Snippet: Aliquots of purified BsaXI RM/S complex from NEB in storage buffer containing 50% glycerol and 25 mM DTT were combined and exchanged into Buffer A (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) during fractionation over a SuperDex600 SEC column to eliminate aggregates and uncomplexed RM and S subunits.

Techniques: