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.

Figure 1. SDS-PAGE analysis of partially purified BisI family enzymes.

Expected enzyme products are marked by “*” (lanes 1–18) from chitin columns following DTT/intein cleavage. M, protein size ladder. See Tables 1, 2, 3 for description of the enzymes.

Figure 2. Bce95I activity assay and run-off sequencing to determine cut sites.

(A) Bce95I digestion of pBRFM, phage λ, XP12, and T4gt DNAs. Bce95I enzyme dilution factors are indicated on the top of each lane. ScaI, ScaI-linearized pBRFM; Fnu4HI, Fnu4HI-digested pBRFM (note: the plasmid is resistant to digestion due to methylation); pBR, Fnu4HI-digested pBR322; “--”, uncut DNA; 2-log, 2-log DNA ladder. (B) DNA run-off sequencing of the Bce95I cleavage site G^m5CTGC and G^m5CAG ^m5CCGC of pBRFM. The up arrow indicates the bottom strand (template) is cleaved. The extra A peak indicates a cut in the bottom strand template (indicated by ↑ arrow). The extra T peak indicates a cut in the top strand (indicated by ↓ arrow). The color-coded sequence traces are: A (green), T (red), C (blue), G (black). The extra A trace (or T on the opposite strand) was added at the end of the cleaved template by the Taq DNA polymerase (template-independent terminal nucleotide transferase activity).

Figure 3. NhoI endonuclease activity assays.

(A) NhoI digestion of phage XP12 (all ^m5C), pBRFM (ScaI-linearized, three ^m5C in G^m5CNG^m5CNGC), and T4gt DNA (^hm5C). Enzyme dilution factors are indicated on the top of each lane. (B) A theoretical digest of pBRFM (NEBcutter) by ScaI and another enzyme cleaving GCNGCNGC (expected sizes in bp: 1168, 966, 828, 515/504/484, 333/297, 153).

Figure 4. Run-off sequencing to determine NhoI cut sites.

DNA run-off sequencing at G^m5CTGC (incubated with NhoI endonuclease, but this site not digested) and G^m5CAG^m5CAGC sites of NhoI-digested plasmid pBRFM (M.Fnu4HI). The G^m5CTGC site indicated by the black bar contains two ^m5C. The G^m5CAG^m5CAGC site indicated by the blue bar contains three ^m5C.

Figure 5. Digestion of ^m5C-modified duplex oligos (GCWGC site) by BisI or NhoI.

The 5′-FAM-labeled top strand contains two ^m5C bases (G^m5CAG^m5C) and the bottom strands contain either two (G^m5CTG^m5C), one (G^m5CTGC, internal C methylated), or no ^m5C bases (GCTGC), respectively. Thus, the annealed oligos contain a total of four, three, or two ^m5C. P1 (20 bp) and P2 (14 bp) are the cleavage products. P3 is a possible top-strand nicked intermediate (NI) due to asymmetric nicking of the top strand. The substrate (sub, 34 bp), P1, and P3 were detected by FAM fluorescence imaging. (A,B). BisI- and NhoI-digested duplex oligos (four, three, or two ^m5C) analyzed on a 15% PAG-urea denaturing gel. C. Partial digestion of the duplex oligos by NhoI and the DNA products were analyzed on a 20% TBE (non-denaturing) gel, stained by SYBR Gold and imaged by fluorescence imaging. The 5-bp dsDNA size marker (Fermentas) and the single-stranded oligos (IDT, 20–100 nt) were used to estimate the size of the cleavage products.

Figure 6. Digestion of DNA duplex oligos with four, three, or two ^m5C in GCWGC by the indicated REases.

Sub, modified duplex DNA substrate; P1, FAM-containing cleavage product (20 bp); NI?, possible top-strand nicking intermediate; ss, single-stranded (top strand) FAM-labeled DNA (34 nt).