Self-generated DNA termini relax the specificity of SgrAl restriction endonuclease

The primary target of SgrAl restriction endonuclease is a multiple sequence of the form 5'-CPu down arrow CCGGPyG. Previous work had indicated that SgrAl must bind two recognition sites simultaneously for catalysis [Bilcock, D. T., Daniels, L. E., Bath, A. J. & Halford, S. E. (1999) J. Biol. Chem. 274, 36379-36386]. In the present study, SgrAl is shown to cleave not only its canonical sequences, but also the sequences 5'-CPuCCGGPy(A,T,C) and 5'-CPuCCGGGG, both referred to as secondary sequences. On plasmid pSK7, SgrAl cleaves secondary sites 26-fold slower than the canonical site. However, the same plasmid, but without the canonical site, is cleaved 200-fold slower. We show that DNA termini generated by cleaving the canonical site for SgrAl assist in the cleavage of secondary sites. The SgrAl-termini in cis with respect to secondary site are markedly preferred over those in trans. The SgrAl-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the SgrAl-termini over substrate, the SgrAl specificity is shown to improve by two orders of magnitude, because of concurrent 10-fold increase in the cleavage of canonical site and 50-fold decrease in the cleavage of secondary sites. The unconventional reaction pathway by which SgrAl utilizes the self-generated DNA termini to cleave its DNA targets has not been observed hitherto among type II restriction endonucleases. Based on our work and previous reports, a pathway of DNA binding and cleavage by the SgrAl restriction endonuclease is proposed.