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Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation.

ABSTRACT: Sequence analysis of the BcnI restriction-modification system from Bacillus centrosporus revealed four open reading frames (bcnIC, bcnIR, bcnIB and bcnIA) that are arranged as two converging collinear pairs. One pair encodes a putative small regulatory protein, C.BcnI, and the restriction endonuclease R.BcnI. The other two gene products are the DNA cytosine-N4 methyltransferases M.BcnIA and M.BcnIB, which differ by circular permutation of conserved sequence motifs. The BcnI methyltransferases are isospecific on double-stranded DNA [methylation specificity CC(C/G)GG], but M.BcnIA can also methylate the target sites in single-stranded DNA. Functional analysis shows that bcnIA is dispensable (bcnIB is capable of protecting the DNA against the in vivo activity of bcnIR); in contrast, no stable clones were obtained if bcnIB alone was deleted from the system. By analogy with the DpnII system, the second methylase M.BcnIA may play a role in the transformation proficiency of its gram-positive host. The interchangeability of homologous elements in the beta class of cytosine-N4 methylases was probed by hybrid formation between M.BcnIB and its closest homolog M.Cfr9I (CCCGGG) employing a novel semi-random strategy combined with selection for catalytic activity. The fusion points in the active hybrids mapped in a narrow region located between sequence motifs X and I. Our data illustrate that recombination of two related sequences by circular permutation may serve as an evolutionary mechanism for creating new specificities of amino MTases.

SUBMITTER: Vilkaitis G

PROVIDER: S-EPMC101829 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation.

Vilkaitis Giedrius G Lubys Arvydas A Merkiene Egle E Timinskas Albertas A Janulaitis Arvydas A Klimasauskas Saulius S

Nucleic acids research 20020401 7

Sequence analysis of the BcnI restriction-modification system from Bacillus centrosporus revealed four open reading frames (bcnIC, bcnIR, bcnIB and bcnIA) that are arranged as two converging collinear pairs. One pair encodes a putative small regulatory protein, C.BcnI, and the restriction endonuclease R.BcnI. The other two gene products are the DNA cytosine-N4 methyltransferases M.BcnIA and M.BcnIB, which differ by circular permutation of conserved sequence motifs. The BcnI methyltransferases ar ...[more]

PMID: 11917015

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Project description:Methylation of cytosine in DNA at position C5 increases the rate of C→T mutations in bacteria and eukaryotes. Methylation at the N4 position, employed by some restriction-modification systems, is not known to increase the mutation rate. Here, I report that a Salmonella enterica Type III restriction-modification system that includes a cytosine-N4 methyltransferase causes an enormous increase in the rate of mutation of the methylated cytosines, which occur at the overlined C in the motif CACC̅GT Mutations consist mainly of C→A transversions, the rate of which is increased ∼500-fold by the restriction-modification system. The rate of C→T transitions is also increased and somewhat exceeds that at C5-methylated cytosines in Dcm sites. Two other Salmonella N4 methyltransferases investigated do not have such dramatic effects, although in one case there is a modest increase in C→A mutations along with an increase in C→T mutations. The sensitivity of the C→A rate to orientation with respect to both DNA replication and transcription is higher at hypermutable sites than at other cytosines, suggesting a fundamental mechanistic difference between hypermutation and ordinary mutation.IMPORTANCE Mutation produces the raw material for adaptive evolution but also imposes a burden because most mutations are deleterious. The rate of mutation at a particular site is affected by a variety of factors. In both prokaryotes and eukaryotes, methylation of C at the C5 position, a naturally occurring DNA modification, greatly increases the rate of C→T mutation. A distinct C modification that occurs in prokaryotes, methylation at N4, is not known to increase mutation rate. Here, I report that a bacterial restriction-modification system, found in some Salmonella bacteria, increases the rate of C→A mutation by a factor of 500 at sites that it methylates at N4. This rate increase is much greater than that caused by C5 methylation. Although fewer than 1 in 1,600 positions analyzed are methylation sites, over 10% of all mutations occur at these sites. Like other examples of extremely high mutation rate, whether naturally occurring or the result of laboratory mutation, this phenomenon may shed light on the mechanism of mutation in general.

Dataset Information

Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation.

Publications

Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation.

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