SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma & Bitinaite, (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, | indicates cut site, R=A,G, Y=C,T) and secondary (CR|CCGGY(A/C/T) and CR|CCGGGG) site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, et al., (2008) Nucleic Acids Res. 36, 5405–5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers (High Molecular Weight Species, HMWS) in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence, that are dependent on the concentration of the DNA bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15–20 s, and is composed of possibly 4–12 DNA bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself, but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA bound SgrAI (i.e. HMWS) are the activated, or activatable, form of the enzyme.

SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200-500 fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that, in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures that are characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating a novel conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N-termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI’s oligomerization and allosteric behavior.

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.

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.

DNA repair is fundamental to genome stability and is found in all three domains of life. However, many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologues, and those present often contain significant differences compared to their eukaryotic homologues. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three dimensional structure were investigated. Both were found to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.