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

Abstract

Sequence analysis of the Bcnl restriction-modification system from Bacillus centrosporus revealed four open reading frames (bcnlC, bcnlR, bcnlB and bcnlA) that are arranged as two converging collinear pairs. One pair encodes a putative small regulatory protein, C.Bcnl, and the restriction endonuclease R.Bcnl. The other two gene products are the DNA cytosine-N4 methyltransferases M.BcnlA and M.BcnlB, which differ by circular permutation of conserved sequence motifs. The Bcnl methyltransferases are isospecific on double-stranded DNA [methylation specificity CC(C/G)GG], but M.BcnlA can also methylate the target sites in single-stranded DNA. Functional analysis shows that bcnlA is dispensable (bcnlB is capable of protecting the DNA against the in vivo activity of bcnlR); in contrast, no stable clones were obtained if bcnlB alone was deleted from the system. By analogy with the Dpnll system, the second methylase M.BcnlA may play a role in the transformation proficiency of its grampositive host. The interchangeability of homologous elements in the β class of cytosine-N4 methylases was probed by hybrid formation between M.BcnlB and its closest homolog M.Cfr9l (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.