Ion Mobility Mass Spectrometry of DNA/SgrAI Nuclease Oligomers

• Main Author: Ma, Xin
• Other Authors: Wysocki, Vicki H.
• Language: en
• Published: The University of Arizona. 2012
• Subjects: protein-DNA complex; SgrAI; Chemistry; collision cross section; ion mobility
• Online Access: http://hdl.handle.net/10150/247282

SgrAI is a restriction endonuclease (ENase) that cuts a long recognition sequence and exhibits self-modulation of cleavage activity and sequence specificity. Previous research has 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 SgrAI:DNA as well as the basic building block of the oligomers, has 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). The oligomers are heterogeneous containing a mixture of species with variable numbers of DBD. The collision cross sections (CCS) of the oligomers were found to have a linear relationship with the number of DBD. Models of the SgrAI/DNA oligomers were constructed and a head-to-tail arrangement was most consistent with the experimental CCS.