The type II restriction endonuclease MvaI has dual specificity

Abstract

The MvaI restriction endonuclease cuts 5'-CC↓AGG-3'/5'-CC↑TGG-3' sites as indicated by the arrows. N4-methylation of the inner cytosines (C(m4)CAGG/C(m4)CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C(m5)C↓GGG/CC(m5)CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CC(m5)C↓GGG/CC(m5)C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the 'normal' activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.

Figure 1.

(A) Digestion of SssI-methylated pUP41 DNA with MvaI and BstNI. Lanes 1, unmethylated pUP41; lanes 2, pUP41 methylated by M.SssI in vitro; lanes 3, pUP41 purified from cells expressing M.SssI; lanes 4, pUP41 purified from cells in which M.SssI production was repressed by glucose; lanes 5, pSTB–MSssI (from uninduced cells). Sizes of fragments that differ between the unmethylated and M.SssI-methylated DNAs are shown in base pairs. The band corresponding to the 543-bp fragment remains visible in the methylated sample because it also contains a comigrating 540-bp fragment. The extra fragments that appear in sub-stoichiometric amounts are indicated by asterisk. M, 1 kb DNA Ladder (Fermentas). (B) Restriction map of the plasmid pUP41. MvaI (spikes) and SmaI (arrows) sites are indicated on the perimeter.

Figure 2.

MvaI cleavage at M.SssI-methylated SmaI sites. (A) Sequencing through the methylated SmaI₂₇₉ site of pUP41 using intact or MvaI-cleaved templates as indicated by the scheme on the left. The terminal adenines, denoted by asterisk, are template-independent additions by Taq polymerase. (B) MvaI cleavage at the canonical recognition sequence and at the M.SssI-methylated SmaI site.

Figure 3.

Comparison of MvaI cleavage rates on the canonical CCWGG/CCWGG and on the M.SssI-methylated SmaI site (CC^m5CGGG/CC^m5CGGG). Digestion of pUP41 plasmid DNA (∼0.5 µg) methylated in vitro by M.SssI. MvaI concentrations are shown above the lanes. M, 1 kb DNA Ladder (Fermentas); -M.SssI, unmethylated plasmid. Appearance of the 1249 and 454 bp fragments indicates cleavage of the two methylated SmaI sites present in pUP41.

Figure 4.

Strand-specific nicking of M.SssI-methylated BcnI sites by MvaI. (A) Possible nicking mechanisms at M.SssI-methylated BcnI sites. (B) Sequencing through the M.SssI-methylated and MvaI-digested BcnI₂₄₁₁ site of pUP41. Asterisk, template-independent addition by the Taq polymerase.

Figure 5.

MvaI digestion of oligonucleotides containing unmethylated, hemimethylated or fully methylated BcnI sites. Electrophoresis of cleavage products in a 10% denaturing polyacrylamide gel. The 30-mer oligonucleotide duplexes contained unmethylated (AK252/254), hemimethylated (AK253/254 and AK252/255) or fully methylated (AK253/255) BcnI sites as shown above the gel. In all duplexes, the G-strand (the strand with G in the central position) was radioactively labeled at the 3′-end. Appearance of a 19-nt fragment indicates cleavage of the G-strand.

Figure 6.

MvaI digestion of the ³²P-labeled 444 bp pUC18 DNA fragment containing an unmethylated BcnI site. (A) Electrophoresis in a 6% denaturing polyacrylamide gel. Lane 1, undigested; lanes 2 and 5, digested with BcnI; lanes 3 and 6, digested with 0.5 U/µl MvaI; lanes 4 and 7, digested with 1.0 U/µl MvaI. Samples in lanes 5, 6 and 7 also contained pUC18 DNA as internal control to test completeness of digestion. Nicking of the G-strand or double-strand cleavage at the single BcnI site produces a 175-nt single-stranded labeled fragment. (B) ImageQuant line graph of lanes 1, 2 and 4. (C) Agarose gel electrophoresis of aliquots of the samples in lanes 5, 6 and 7. pUC, pUC18 completely digested with BcnI or MvaI. M, 1 kb DNA Ladder (Fermentas).