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.

Here, we investigate an unusual antiviral mechanism developed in the bacterium Streptomyces griseus. SgrAI is a type II restriction endonuclease that forms run-on oligomer filaments when activated and possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust antiphage activity of wild-type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against damaging DNA cleavage activity of activated SgrAI. IMPORTANCE This work is motivated by an interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted, and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzyme’s activation compared to that of other known mechanisms, as well as to better localize its action to molecules of interest, such as invading phage DNA.

Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI-DNA complexes into the run-on oligomer filament is relatively slow (3-4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI-DNA. The disassembly step involving dissociation of complexes of SgrAI-DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI-DNA complexes and faster than dissociation of SgrAI-DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.

Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.

Infection with human parvovirus B19 (B19V) has been associated with a myriad of illnesses, including erythema infectiosum (Fifth disease), hydrops fetalis, arthropathy, hepatitis, and cardiomyopathy, and also possibly the triggering of any number of different autoimmune diseases. B19V NS1 is a multidomain protein that plays a critical role in viral replication, with predicted nuclease, helicase, and gene transactivation activities. Herein, we investigate the biochemical activities of the nuclease domain (residues 2–176) of B19V NS1 (NS1-nuc) in sequence-specific DNA binding of the viral origin of replication sequences, as well as those of promoter sequences, including the viral p6 and the human p21, TNFα, and IL-6 promoters previously identified in NS1-dependent transcriptional transactivation. NS1-nuc was found to bind with high cooperativity and with multiple (five to seven) copies to the NS1 binding elements (NSBE) found in the viral origin of replication and the overlapping viral p6 promoter DNA sequence. NS1-nuc was also found to bind cooperatively with at least three copies to the GC-rich Sp1 binding sites of the human p21 gene promoter. Only weak or nonspecific binding of NS1-nuc to the segments of the TNFα and IL-6 promoters was found. Cleavage of DNA by NS1-nuc occurred at the expected viral sequence (the terminal resolution site), but only in single-stranded DNA, and NS1-nuc was found to covalently attach to the 5′ end of the DNA at the cleavage site. Off-target cleavage by NS1-nuc was also identified.