title: Probing the run-on oligomer of activated SgrAI bound to DNA.
abstract: SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3'-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America.
authors.forename: Santosh
authors.initials: S
authors.lastname: Shah
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Sanchez
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America; Genetics Graduate Program, University of Arizona, Tucson, AZ 85721, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Stewart
authors.affiliation: Department of Chemistry, University of Liverpool, Liverpool, United Kingdom, L69 7ZD, United States of America.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Piperakis
authors.affiliation: Department of Chemistry, University of Liverpool, Liverpool, United Kingdom, L69 7ZD, United States of America.
authors.forename: Richard
authors.initials: R
authors.lastname: Cosstick
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America.
authors.forename: Claire
authors.initials: C
authors.lastname: Nichols
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America.
authors.forename: Chad K
authors.initials: CK
authors.lastname: Park
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America; Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH 43210, United States of America.
authors.forename: Xin
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America; Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH 43210, United States of America.
authors.forename: Vicki
authors.initials: V
authors.lastname: Wysocki
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, United States of America.
authors.forename: Jurate
authors.initials: J
authors.lastname: Bitinaite
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, United States of America.
authors.forename: Nancy C
authors.initials: NC
authors.lastname: Horton
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D013379:Substrate Specificity
chemical_list: C465503:3'-S-phosphorothiolate; D010710:Phosphates; D004247:DNA; C064435:endodeoxyribonuclease SgrAI; D015252:Deoxyribonucleases, Type II Site-Specific