title: Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes.
abstract: The methylation-dependent restriction endonuclease (REase) BisI (G(m5)C ↓ NGC) is found in Bacillus subtilis T30. We expressed and purified the BisI endonuclease and 34 BisI homologs identified in bacterial genomes. 23 of these BisI homologs are active based on digestion of (m5)C-modified substrates. Two major specificities were found among these BisI family enzymes: Group I enzymes cut GCNGC containing two to four (m5)C in the two strands, or hemi-methylated sites containing two (m5)C in one strand; Group II enzymes only cut GCNGC sites containing three to four (m5)C, while one enzyme requires all four cytosines to be modified for cleavage. Another homolog, Esp638I cleaves GCS ↓ SGC (relaxed specificity RCN ↓ NGY, containing at least four (m5)C). Two BisI homologs show degenerate specificity cleaving unmodified DNA. Many homologs are small proteins ranging from 150 to 190 amino acid (aa) residues, but some homologs associated with mobile genetic elements are larger and contain an extra C-terminal domain. More than 156 BisI homologs are found in >60 bacterial genera, indicating that these enzymes are widespread in bacteria. They may play an important biological function in restricting pre-modified phage DNA.
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Shuang-Yong
authors.initials: SY
authors.lastname: Xu
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Pernelle
authors.initials: P
authors.lastname: Klein
authors.affiliation: SibEnzyme Ltd., 2/12 Ak, Timakov Street, Novosibirsk 630117, Russia.
authors.forename: Sergey Kh
authors.initials: SKh
authors.lastname: Degtyarev
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Roberts
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D015253:Deoxyribonucleases, Type I Site-Specific / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D000072417:Protein Domains; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D015253:Deoxyribonucleases, Type I Site-Specific