DNA methyltransferases from Neisseria meningitidis and Neisseria gonorrhoeae FA1090 associated with mismatch nicking endonucleases

Abstract

The genes encoding the DNA methyltransferases M.NmeDI and M.NmeAI from Neisseria meningitidis associated with the genes encoding putative Vsr endonucleases were overexpressed in Escherichia coli. The enzymes were purified to apparent homogeneity on Ni-NTA agarose columns, yielding proteins of 49+/-1 kDa and 39.6+/-1 kDa, respectively, under denaturing conditions. M.NmeDI recognizes the degenerate sequence 5'-RCCGGB-3'. It methylates the first 5' cytosine residue on both strands within the core sequence CCGG. The enzyme shows higher affinity with the hemimethylated degenerate sequence than with the unmethylated degenerate sequence. Comparison of the amino acid sequence of the target-recognizing domain of M.NmeDI with the closest neighbours recognizing the sequence 5'-RCCGGY-3' showed the presence of the homologous domain and an additional domain that may be responsible for recognizing the degenerate sequence. M.NmeAI recognizes the sequence 5'-CCGG-3' and methylates the second 5' cytosine residue on both DNA strands. In Neisseria gonorrhoeae strain FA1090 the homologues of these ORFs are truncated due to a variety of mutations.

An orphan DNA (cytosine-5-)-methyltransferase in Vibrio cholerae

“…Further analysis of these refractory sites reveal that they fall within the sequence 59-RCCGGY-39. Failure of both HpaII and MspI to cleave at 59-RCCGGY-39 sites suggested that the first 59 C within the sequence 59-RCCGGY-39 is methylated by M.Vch, since methylation of only this cytosine blocks both MspI and HpaII cleavage (Kwiatek et al, 2004;Roberts et al, 2005). When plasmid pBR322 isolated from strain CM319 (DvchM) was digested with HpaII or MspI, all potential sites were completely cleaved, yielding a pattern similar to that obtained in HpaII and MspI digests of pBR322 DNA from E. coli (Figs 4a and b, lanes 1 and 3), indicating absence of m5C in the recognition sites.…”

The Type IIB restriction endonucleases

“…NmeDI provides a further example of a two-polypeptide Type IIB R-M system, in this case two entirely separate proteins: the NmeDI REase [22] and the NmeDI MTase [29]. The NmeDI system is thus organized like a conventional two-protein Type II system [3], unlike the multisubunit creatures of the Type I and other Type IIB systems.…”

“…The REase appears from gel filtration to be a tetramer, but many Type II REases are tetramers, particularly those in the Type IIF group that cleave two recognition sites concertedly (Table 1 [30,31]). Even though the NmeDI REase makes doublestrand breaks on both sides of its site and so has to be classified as a IIB system, it is distinctive in numerous respects: it has a continuous recognition sequence, as opposed to the discontinuous bipartite sites for all other Type IIB systems (Table 2); the NmeDI MTase modifies cytosine residues [29], whereas all other Type IIB enzymes act at adenine residues; it alone leaves 5 rather than 3 single-strand extensions.…”

Neisseria gonorrhoeae FA1090 Carries Genes Encoding Two Classes of Vsr Endonucleases

“…The central enzyme of this pathway is Vsr, an endonuclease whose coding sequence overlaps the gene for M.EcoKDcm, an m5C methyltransferase (m5C-MTase) (19,23). In genomes of other bacteria, the vsr genes are invariably associated with genes coding for m5C-MTases (3,16,20). The Vsr endonucleases that accompany m5C-MTases are believed to exhibit sequence specificity based on the recognition sequence of the accompanying MTase.…”

“…Furthermore, we were only able to identify two potential Vsr endonucleases. While the genes encoding both of these proteins appear to be linked to restriction-modification system genes in a variety of gonococcal strains, these systems appear to be inactive (16). To understand the biochemical basis of VSP repair in the Neisseriaceae, we studied the properties of Vsr endonucleases from N. gonorrhoeae FA1090.…”