Structural analysis of activated SgrAI-DNA oligomers using ion mobility mass spectrometry

Xin Ma, Santosh Shah, Mowei Zhou, Chad K. Park, Vicki H. Wysocki, Nancy C Horton

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)4373-4381
Number of pages9
JournalBiochemistry
Volume52
Issue number25
DOIs
StatePublished - Jun 25 2013

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Oligomers
Structural analysis
Mass spectrometry
Mass Spectrometry
Ions
DNA
DNA Cleavage
Dimers
DNA Restriction Enzymes
DNA sequences
Observation
Stoichiometry
Ionization
Enzymes
Modulation
Molecules
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural analysis of activated SgrAI-DNA oligomers using ion mobility mass spectrometry. / Ma, Xin; Shah, Santosh; Zhou, Mowei; Park, Chad K.; Wysocki, Vicki H.; Horton, Nancy C.

In: Biochemistry, Vol. 52, No. 25, 25.06.2013, p. 4373-4381.

Research output: Contribution to journalArticle

Ma, Xin ; Shah, Santosh ; Zhou, Mowei ; Park, Chad K. ; Wysocki, Vicki H. ; Horton, Nancy C. / Structural analysis of activated SgrAI-DNA oligomers using ion mobility mass spectrometry. In: Biochemistry. 2013 ; Vol. 52, No. 25. pp. 4373-4381.
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