Self-generated DNA termini relax the specificity of SgrAI restriction endonuclease.
The primary target of SgrAI restriction endonuclease is a multiple sequence of the form 5'-CPu/CCGGPyG. 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.
Bitinaite
Jurate
J
New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA. bitinait@neb.com
Schildkraut
Ira
I
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; C100902:endodeoxyribonuclease SapI; C064435:endodeoxyribonuclease SgrAI; D015252:Deoxyribonucleases, Type II Site-Specific