Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

Lyumkis, Dmitry; Talley, Heather; Stewart, Andrew M.; Shah, Santosh; Park, Chad K.; Tama, Florence; Potter, Clinton S.; Carragher, Bridget; Horton, Nancy C.

doi:10.1016/j.str.2013.08.012

Cited by 25 publications

(86 citation statements)

References 56 publications

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“…Lastly, the LC and RGG-ZnF-RGG domains interact with each other in trans in an RNA-dependent manner (53). Analogous allosteric regulation of nucleic acid binding proteins that triggers self-assembly has been proposed for the bacterial SgrAI and eukaryotic IreI proteins (75,76).…”

Section: Fet Protein Interactionsmentioning

confidence: 99%

Biochemical Properties and Biological Functions of FET Proteins

Schwartz

Cech

Parker

2015

Annu. Rev. Biochem.

193

240

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Members of the FET protein family, consisting of FUS, EWSR1, and TAF15, bind to RNA and contribute to the control of transcription, RNA processing, and the cytoplasmic fates of messenger RNAs in metazoa. FET proteins can also bind DNA, which may be important in transcription and DNA damage responses. FET proteins are of medical interest because chromosomal rearrangements of their genes promote various sarcomas and because point mutations in FUS or TAF15 can cause neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. Recent results suggest that both the normal and pathological effects of FET proteins are modulated by low-complexity or prion-like domains, which can form higher-order assemblies with novel interaction properties. Herein, we review FET proteins with an emphasis on how the biochemical properties of FET proteins may relate to their biological functions and to pathogenesis.

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Section: Fet Protein Interactionsmentioning

confidence: 99%

Biochemical Properties and Biological Functions of FET Proteins

Schwartz

Cech

Parker

2015

Annu. Rev. Biochem.

193

240

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“…II RE SgrAI from Streptomyces griseus led us to propose a new mechanism of enzyme regulation involving filament formation (7,8).…”

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confidence: 99%

“…At approximately the same time, large-scale screens for protein localization using fluorescence microscopy showed unexpectedly that many enzymes formed filaments in response to particular metabolic conditions or other stimuli in cells (11,(13)(14)(15). The term "run-on oligomer" (ROO) filament is used here to describe an assembly of an enzyme into a filament by the successive addition of enzymes at either end, and which, in principle, could extend indefinitely (8,16). ROO filament formation by SgrAI was first proposed in 2010 based on behavior in analytical ultracentrifugation and native gels (7) and subsequently using ion-mobility mass spectrometry (17).…”

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confidence: 99%

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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“…However, assemblies containing many more DBDs were shown by analytical ultracentrifugation and ionmobility mass spectrometry 10,19 . Negative stain EM revealed filaments of varied lengths with left-handed helical symmetry that we call run-on oligomers (ROO) 20 .…”

Section: Introductionmentioning

confidence: 99%

“…The ROO filament structure was previously resolved to ~nanometer resolution by cryo-EM and helical reconstruction, that revealed left-handed helical symmetry with approximately 4 DBDs per turn 20 . This structure inspired a low-resolution mechanistic model for the enzymatic behavior of SgrAI, wherein binding to primary site DNA induces a conformational change that favors ROO filament formation, which in turn stabilizes the activated enzyme state capable of accelerated DNA cleavage ( Fig.…”

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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Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

Cited by 25 publications

References 56 publications

Biochemical Properties and Biological Functions of FET Proteins

Biochemical Properties and Biological Functions of FET Proteins

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

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