In vivo restriction endonuclease activity of the Anabaena PCC 7120 XisA protein in Escherichia coli

Abstract

Anabaena PCC 7120 genome contains three elements, which get excised out during late stages of heterocyst differentiation by a site-specific recombination process. The XisA protein, which excises the nifD element, shows sequence homology with the integrase family of tyrosine recombinase. The 11 bp target site of XisA C GGAGTAA TCC contains a 3 bp inverted repeat. Here, we report restriction endonuclease activity of XisA by specific loss of plasmids containing single or double target sites. The pMX25 plasmid containing two target sites demonstrated endonuclease activity proportional to excision frequency. Different plasmid substrates containing one base pair mutation in the inverted repeat of the target site were monitored for endonuclease activity. Mutation of A4C retained endonuclease activity, while other modifications lost endonuclease activity. The presence of an additional copy of the target site enhanced endonuclease activity. These results suggest that the XisA protein could be an IIE type of restriction endonuclease in addition to being a recombinase.