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.
Highlights
SgrAI, a type II restriction endonuclease, appears to be a member of a growing list of enzymes that form filaments or run-on oligomers with modulated enzymatic activities [1]. This list currently includes members such as IRE1, an RNA splicing nuclease/ protein kinase involved in the unfolded protein response [2], acetyl-CoA carboxylase (ACC)[3], and CTP synthase (CTPS) [4]
Previous studies show that activation of SgrAI was possible with either intact or with precleaved primary site DNA added to the DNA cleavage assays, it was not known if the uncleaved DNA itself was the activator, or if it must be cleaved first to become the activator [5,6]
To test whether cleavage of the primary site DNA is a prerequisite for its ability to activate SgrAI, a noncleavable oligonucleotide with a 3’-S-phosphorothiolate substitution (22-13’S) at the site of cleavage by SgrAI was prepared (Materials and Methods, flanking DNA as 40–1 and 40-2A (Fig 1))
Summary
SgrAI, a type II restriction endonuclease, appears to be a member of a growing list of enzymes that form filaments or run-on oligomers with modulated (activated or inhibited) enzymatic activities [1] This list currently includes members such as IRE1, an RNA splicing nuclease/ protein kinase involved in the unfolded protein response [2], acetyl-CoA carboxylase (ACC)[3], and CTP synthase (CTPS) [4]. Unlike amyloid, these run-on oligomers are transient, reversible, and activate (or inhibit) enzyme activity. The proposed biological roles for run-on oligomerization by the different enzymes include rapid activation (or inactivation), storage of inactive enzyme, increase of substrate affinity through larger binding surfaces, and uniquely to SgrAI, sequestration of activated SgrAI on invading phage DNA (which protects the host bacterial genome from secondary site cleavage by activated SgrAI) [8,9,10]
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