Probing the Run-On Oligomer of Activated SgrAI Bound to DNA

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.

Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease

“…To elucidate the molecular determinants of SgrAI activation, we sought to resolve the ROO filament to near-atomic resolution. We prepared ROO filaments from purified, recombinant, his-tagged wild type SgrAI 14 bound to a 40-bp oligonucleotide DNA containing a pre-cleaved primary site sequence (PC DNA, see Methods). Cryo-EM micrographs revealed a large variety of differently-sized filaments, as expected for a ROO ( Fig.…”

“…Furthermore, DNA bending allows for interactions with residues 56-57 and 127-134 of the neighboring SgrAI, without steric conflicts. These residues have been shown to be important for enzyme activation, presumably by stabilizing the ROO filament 14 . Overall, the structural rearrangements observed within the filamentous SgrAI ROO (Movie S1) prevent unfavorable clashes and promote interactions between neighboring DBDs.…”

“…This gives an equilibrium constant that shows that the T state is favored over the R state by 470-fold (0.8 s -1 /0.0017 s -1 ), corresponding to a free energy difference of −3.6 kcal/mol (at 25°C). Since SgrAI cleaves secondary site DNA much more slowly 14 , the T state is favored by this complex to a greater extent, giving −4.9 kcal/mol ( Fig. 5D ).…”

“…SgrAI is a sequence-specific type II DNA restriction endonuclease that is allosterically activated by the same substrate DNA. A primary recognition sequence (CR|CCGGYG, where R=A or G and Y=C or T, | denotes cleavage site) activates the enzyme by over 200-fold, which in turn also relaxes the enzyme’s DNA sequence specificity, resulting in the cleavage of an additional 14 secondary sequences (C C CCGGYG or X RCCGGY G, where X=A,G, or T) up to 1000-fold faster 10–14 . In the absence of DNA, SgrAI is a homodimer composed of two 37 kDa chains, each with a single active site 15–18 .…”

“…1B ). This explains why cleavage of secondary site sequences is negligible unless a primary site is present – in sufficient concentration or on the same contiguous DNA – to promote ROO filament assembly 10,12,14 . Filaments formed by SgrAI bound to primary site DNA will drive ROO assembly, incorporating DBD-containing secondary sites, activating SgrAI for DNA cleavage on both primary and secondary sites, and thereby expanding the enzyme’s DNA sequence specificity ( Fig.…”