Probing the run-on oligomer of activated SgrAI bound to DNA

Santosh Shah, Jonathan Sanchez, Andrew Stewart, Michael M. Piperakis, Richard Cosstick, Claire Nichols, Chad K. Park, Xin Ma, Vicki Wysocki, Jurate Bitinaite, Nancy C. Horton

Research output: Contribution to journalArticle

8 Scopus citations

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 runon 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.

Original languageEnglish (US)
Article numbere0124783
JournalPloS one
Volume10
Issue number4
DOIs
StatePublished - Apr 16 2015

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Shah, S., Sanchez, J., Stewart, A., Piperakis, M. M., Cosstick, R., Nichols, C., Park, C. K., Ma, X., Wysocki, V., Bitinaite, J., & Horton, N. C. (2015). Probing the run-on oligomer of activated SgrAI bound to DNA. PloS one, 10(4), [e0124783]. https://doi.org/10.1371/journal.pone.0124783