title: Structural analysis of activated SgrAI-DNA oligomers using ion mobility mass spectrometry.
abstract: SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits self-modulation of DNA cleavage activity and sequence specificity. Previous studies have shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of the SgrAI-DNA complex as well as the basic building block of the oligomers have not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI-DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD) with one SgrAI dimer bound to two precleaved duplex DNA molecules each containing one-half of the SgrAI primary recognition sequence. The oligomers contain variable numbers of DBDs with as many as 19 DBDs. Observation of the large oligomers shows that nanoelectrospray ionization (nano-ESI) can preserve the proposed activated form of an enzyme. Finally, the collision cross section of the SgrAI-DNA oligomers measured by IM-MS was found to have a linear relationship with the number of DBDs in each oligomer, suggesting a regular, repeating structure.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States.
authors.forename: Xin
authors.initials: X
authors.lastname: Ma
authors.forename: Santosh
authors.initials: S
authors.lastname: Shah
authors.forename: Mowei
authors.initials: M
authors.lastname: Zhou
authors.forename: Chad K
authors.initials: CK
authors.lastname: Park
authors.forename: Vicki H
authors.initials: VH
authors.lastname: Wysocki
authors.forename: Nancy C
authors.initials: NC
authors.lastname: Horton
mesh_terms: D018360:Crystallography, X-Ray; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D021241:Spectrometry, Mass, Electrospray Ionization; D013305:Streptomyces griseus / Q000201:enzymology
chemical_list: C064435:endodeoxyribonuclease SgrAI; D015252:Deoxyribonucleases, Type II Site-Specific