Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

Abstract

The primary target of SgrAI restriction endonuclease is a multiple sequence of the form 5 -CPu2CCGGPyG. Previous work had indicated that SgrAI 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, SgrAI 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, SgrAI 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 SgrAI assist in the cleavage of secondary sites. The SgrAI-termini in cis with respect to secondary site are markedly preferred over those in trans. The SgrAI-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the SgrAI-termini over substrate, the SgrAI 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 SgrAI 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 SgrAI restriction endonuclease is proposed.

Kinetic Analysis of the Coordinated Interaction of SgrAI Restriction Endonuclease with Different DNA Targets

“…However, as revealed by the binding data, neither the truncated phosphodiester bonds nor the 5Ј phosphate groups seem to be of a great importance for SgrAI binding. These results are in agreement with our previous work (35) that shows that the dephosphorylated canonical site cleavage products are as capable of assisting in secondary site cleavage as those flanked with 5Ј phosphates.…”

“…The values are shown as mean Ϯ range. DNA termini generated by SgrAI cleavage at the canonical site assist to a great degree in the cleavage of cis-oriented secondary sites (35). These data imply that SgrAI cleavage products may act as specific activators in the SgrAI-catalyzed reaction.…”

“…To evaluate the effect of the specific duplex on the cleavage rates at a secondary site (CACCGGCT), the molar ratio between the substrate and the specific duplex was maintained at 1:4, because at higher concentrations of specific duplex, an inhibitory effect on secondary site cleavage was previously observed (35). In the presence of the specific duplex, a nonlinear increase in the initial velocities of secondary site cleavage was encountered at all ranges of substrate concentrations.…”

“…The sequence of the oligonucleotide duplex flanked by the nonspecific termini was identical, except that it carried 5Ј-GGCC ss extensions. The cleavage reactions in the presence of the oligonucleotide duplex were performed as described above, except the oligonucleotide duplex was added at a 50-fold molar excess over the substrate with the canonical site and at a 4-fold molar excess over the substrate with the secondary site (35).…”

“…However, in contrast to the SfiI-like enzymes, SgrAI was found to exist as a homodimer in a solution (34). Furthermore, the SgrAI endonuclease has been found to cleave effectively at a subset of less-specific sequences, 5Ј-CPuCCGGPy(A/T/C) and 5Ј-CPuCCGGGG, referred to as secondary sites (35). DNA termini generated by cleaving at the SgrAI canonical site are a pre-requisite for efficient cleavage at secondary sites.…”