The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament

Park, Chad K.; Sanchez, Jonathan L.; Barahona, Claudia; Basantes, L. Emilia; Sanchez, Juan; Hernández, Christian; Horton, Nancy C.

doi:10.1074/jbc.ra118.003680

Cited by 9 publications

(47 citation statements)

References 41 publications

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“…In order to simulate the reaction in vivo, we estimate concentrations of SgrAI and DNA in the cell and also estimate the local concentration of SgrAI when bound to sites on the same contiguous DNA molecule. Using these concentrations, and the kinetic model and rate constants derived in prior work (24,25), we discover that while the relatively low association rate constant of SgrAI/DNA complexes into the ROO filament does in fact limit the rate of reaction, it importantly is also the source of the proposed sequestration effect that protects the host genome from the potentially damaging activity of SgrAI. This is because it limits ROO filament formation within the cell to only those SgrAI enzymes bound to the same DNA molecule, meaning in vivo SgrAI would cleave only invading phage DNA and not the host genome.…”

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confidence: 90%

“…Being a relatively newly described enzyme mechanism, several fundamental questions are of interest, including (i) how the ROO filament accelerates the formation of the product of the reaction (i.e., cleaved DNA) without trapping it in the filament, (ii) whether or not the assembly and/or the disassembly of the ROO limits the overall rate of reaction, (iii) what the growth and dissolution mechanism of the ROO filament is (e.g., from the ends only or occurring anywhere in the filament), and (iv) what are the special advantages, if any, of the ROO filament mechanism (over more conventional mechanisms) that evolved due to the particular biological niche of SgrAI. The first three issues were addressed in prior work (24,25), which showed that ROO filament assembly is rate limiting in in vitro reactions at low concentrations of SgrAI and DNA. It was also found that DNA cleavage is rapid in the ROO filament, faster than dissociation of the ROO filament, making the reaction pathway efficient, since DNA cleavage is much more likely with each addition to the ROO filament prior to its dissociation.…”

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confidence: 99%

“…Using kinetic modeling and rate constants derived and determined from prior work (24,25), we next simulate the in vivo kinetics of SgrAI activity. We also build an alternative, non-ROO model to use side by side in simulations in order to discover any advantages or limitations inherent to the ROO filament mechanism.…”

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confidence: 99%

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The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

Barahona

Basantes

Tompkins

et al. 2019

J Virol

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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.

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The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

Barahona

Basantes

Tompkins

et al. 2019

J Virol

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“…In the case of SgrAI, an extensive kinetic study has recently been performed showing that association of SgrAI/DNA complexes into the ROO filament is the rate determining step under most conditions, and is overcome only through high enzyme and DNA concentrations (obtainable in vitro ). In a biological context, and owing to local concentration effects, filament formation is expected to occur when two cleavage sites reside on the same contiguous DNA 21–23 . This effect is significant and is responsible for sequestering SgrAI activity on phage DNA and away from the host genome 23 .…”

Section: Resultsmentioning

confidence: 99%

“…A full kinetic analysis of SgrAI-mediated cleavage of primary site DNA enabled the estimation of individual rate constants for the major steps in the reaction pathway, such as DBD association into the ROO filament, cleavage of DNA within the filament, DBD dissociation from the ROO filament, and dissociation of cleaved DNA from SgrAI 21,22 . These studies show that assembly of DBDs into the ROO filament is rate limiting when recognition sites are on separate DNA molecules, but fast when on the same contiguous DNA.…”

Section: Introductionmentioning

confidence: 99%

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

Polley

Lyumkis

Horton

2019

Preprint

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23 metal ion mechanism, protein filament 24 25 26 ABSTRACT 1 2Filament or run-on oligomer formation by enzymes is increasingly recognized as an important 3 phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an 4 allosterically regulated type II restriction endonuclease that forms run-on oligomeric (ROO) 5 filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present 6 the 3.5 Å cryo-electron microscopy structure of the ROO filament of SgrAI bound to a mimic of 7 cleaved primary site DNA and Mg 2+ . Large conformational changes stabilize a second metal ion 8 cofactor binding site within the catalytic pocket and facilitate assembling a higher-order enzyme 9 form that is competent for rapid DNA cleavage. The structural changes illuminate the mechanistic 10 origin of hyper-accelerated DNA cleavage activity within the filamentous SgrAI form. An analysis of 11 the protein-DNA interface and the stacking of individual nucleotides reveals how indirect DNA 12 readout within filamentous SgrAI enables recognition of substantially more nucleotide sequences 13 than its low-activity form, thereby expanding DNA sequence specificity. Together, substrate DNA 14 binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and 15 modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the 16 specialized functions of bacterial innate immunity in rapid defense against invading phage DNA 17 without causing damage to the host DNA. 18 19 20 2 Filament formation by non-cytoskeletal enzymes is a newly appreciated phenomenon. Although first 3 shown in vitro for the metabolic enzymes acetyl-CoA carboxylase and phosphofructokinase in the 1970s 1-3 , it 4 was not until 40 years later that filament formation was demonstrated to modulate enzyme activity under 5 physiological conditions 4,5 . At around the same time, filament formation was surprisingly discovered for the 6 unfolded protein response RNase/kinase Ire1 6 and for numerous other enzymes previously unknown to form 7 such assemblies 7-9 . Independently, filament formation was proposed to explain the unusual biophysical and 8 biochemical activities seen in the type II restriction endonuclease SgrAI 10 .

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Structures, functions, and mechanisms of filament forming enzymes: a renaissance of enzyme filamentation

2019

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Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI system are also highlighted.3 Contents INTRODUCTION…………………………………………………………..4 STRUCTURALLY CHARACTERIZED ENZYME FILAMENTS…….5

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The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament

Cited by 9 publications

References 41 publications

The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

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

Structures, functions, and mechanisms of filament forming enzymes: a renaissance of enzyme filamentation

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