About

Nancy Horton is a professor whose research interests include the biological role and regulatory mechanisms of enzyme filamentation, rapid stopped flow enzyme kinetics, and cryo-electron microscopy. She has a background in biochemistry, holding a Ph.D. in Chemistry from UCSB under the mentorship of Prof. Jacob Israelashvili, and a B.S. in Chemical Engineering from the University of Delaware. Her work involves understanding enzyme behavior at a molecular level, contributing to the broader field of biochemistry and molecular biology.

Research topics

• Biochemistry
• Chemistry
• Biology
• Biophysics
• Computational biology
• Chromatography
• Cell biology

Selected publications

• Structures, mechanisms, and kinetic advantages of the SgrAI filament forming mechanism

  Critical Reviews in Biochemistry and Molecular Biology · 2024-11-01

  review1st authorCorresponding

  host genome.

  PublisherDOI

• Crosstalk between co‐assembling filamentous enzymes

  BioEssays · 2024-06-08

  article1st authorCorresponding

  The author declares no conflicts of interest.

  PublisherDOI

• Two-metal ion mechanism of DNA cleavage by activated, filamentous SgrAI

  Journal of Biological Chemistry · 2024-07-14 · 1 citations

  articleOpen accessSenior authorCorresponding

  Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes. Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes. Enzyme filamentation involves the polymerization of multiple copies of the same protein into long linear, helical, or tubular structures. Filamentation has been observed for decades, yet only recently has it been generally acknowledged to be a widespread mechanism of enzyme regulation (1Park C.K. Horton N.C. Structures, functions, and mechanisms of filament forming enzymes: a Renaissance of enzyme filamentation.Biophys. Rev. 2019; 11: 927-994Crossref PubMed Scopus (60) Google Scholar, 2Park C.K. Horton N.C. Novel insights into filament-Forming enzymes.Nat. Rev. Mol. Cell Biol. 2020; 21: 1-2Crossref PubMed Scopus (11) Google Scholar, 3Lynch E.M. Kollman J.M. Webb B.A. Filament Formation by metabolic enzymes-a new twist on regulation.Curr. Opin. Cell Biol. 2020; 66: 28-33Crossref PubMed Scopus (31) Google Scholar, 4Simonet J.C. Burrell A.L. Kollman J.M. Peterson J.R. Freedom of assembly: metabolic enzymes Come Together.Mol. Biol. Cell. 2020; 31: 1201-1205Crossref PubMed Scopus (1) Google Scholar, 5Prouteau M. Loewith R. Regulation of cellular metabolism through phase separation of enzymes.Biomolecules. 2018; 8: 160-173Crossref PubMed Scopus (60) Google Scholar). To date, over 30 enzymes from diverse biochemical pathways and from organisms representing all domains of life have been shown to form filamentous assemblies (1Park C.K. Horton N.C. Structures, functions, and mechanisms of filament forming enzymes: a Renaissance of enzyme filamentation.Biophys. Rev. 2019; 11: 927-994Crossref PubMed Scopus (60) Google Scholar, 2Park C.K. Horton N.C. Novel insights into filament-Forming enzymes.Nat. Rev. Mol. Cell Biol. 2020; 21: 1-2Crossref PubMed Scopus (11) Google Scholar). A subset of these enzymes have been subjected to detailed studies to determine how filamentation affects enzyme activity (6Lyumkis D. Horton N.C. The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease Sgrai.Biochem. Soc. Trans. 2022; 50: 1703-1714Crossref PubMed Scopus (0) Google Scholar, 7Hvorecny K.L. Kollman J.M. Greater than the Sum of parts: mechanisms of metabolic regulation by enzyme filaments.Curr. Opin. Struct. Biol. 2023; 79102530Crossref PubMed Scopus (11) Google Scholar). Many of the same enzymes known to form filaments in vitro form in that are by known as (1Park C.K. Horton N.C. Structures, functions, and mechanisms of filament forming enzymes: a Renaissance of enzyme filamentation.Biophys. Rev. 2019; 11: 927-994Crossref PubMed Scopus (60) Google Scholar, 2Park C.K. Horton N.C. Novel insights into filament-Forming enzymes.Nat. Rev. Mol. Cell Biol. 2020; 21: 1-2Crossref PubMed Scopus (11) Google Scholar, 5Prouteau M. Loewith R. Regulation of cellular metabolism through phase separation of enzymes.Biomolecules. 2018; 8: 160-173Crossref PubMed Scopus (60) Google Scholar, M. and of Rev. Cell Mol. Biol. PubMed Scopus Google Scholar, R. M. M. of metabolic enzymes into assemblies PubMed Scopus Google Scholar, of metabolic enzymes in PubMed Scopus Google Scholar, The and its filamentation and of metabolic Rev. Cell Biol. PubMed Scopus Google Scholar, D. R. A for metabolic enzyme structures reveals of the metabolic Biol. Cell. 2019; PubMed Scopus Google Scholar, of in PubMed Scopus Google Scholar). In a direct between and enzyme has been shown M. Loewith R. Regulation of cellular metabolism through phase separation of enzymes.Biomolecules. 2018; 8: 160-173Crossref PubMed Scopus (60) Google Scholar, B.A. Kollman J.M. The enzyme into Cell Biol. PubMed Scopus Google Scholar). The of these cellular is have been suggested filamentation to the of cellular or of the of active enzyme in the In the have been suggested to phase containing enzymes from a biochemical to within pathways D. R. A for metabolic enzyme structures reveals of the metabolic Biol. Cell. 2019; PubMed Scopus Google Scholar, B.A. Kollman J.M. The enzyme into Cell Biol. PubMed Scopus Google Scholar, R. Filamentation of the is for substrate and enzymatic 2020; 11: PubMed Scopus Google Scholar, J.C. enzymes to to support PubMed Scopus Google Scholar). enzymatic specificity or (1Park C.K. Horton N.C. Structures, functions, and mechanisms of filament forming enzymes: a Renaissance of enzyme filamentation.Biophys. Rev. 2019; 11: 927-994Crossref PubMed Scopus (60) Google Scholar, 2Park C.K. Horton N.C. Novel insights into filament-Forming enzymes.Nat. Rev. Mol. Cell Biol. 2020; 21: 1-2Crossref PubMed Scopus (11) Google Scholar, 7Hvorecny K.L. Kollman J.M. Greater than the Sum of parts: mechanisms of metabolic regulation by enzyme filaments.Curr. Opin. Struct. Biol. 2023; 79102530Crossref PubMed Scopus (11) Google Scholar). of the enzymes that forms filamentous assemblies is the type II restriction endonuclease SgrAI an A or or cleavage of a (6Lyumkis D. Horton N.C. The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease Sgrai.Biochem. Soc. Trans. 2022; 50: 1703-1714Crossref PubMed Scopus (0) Google Scholar, M. a Novel restriction endonuclease from the sequence PubMed Google Scholar). The sequence at the second and which to a of different in DNA that are known as or that enzyme SgrAI known as or with the and or DNA the specificity of restriction PubMed Scopus Google Scholar, C.K. Horton N.C. of DNA cleavage by of PubMed Scopus Google Scholar, R. the of to Scopus Google Scholar). to with the sequence and in the filamentous SgrAI its and and than in the C.K. Horton N.C. of DNA cleavage by of PubMed Scopus Google Scholar, R. the of to Scopus Google Scholar, M. C.K. Horton N.C. of using PubMed Scopus Google Scholar). SgrAI and in a known as the DNA SgrAI C.K. Horton N.C. of DNA cleavage by of PubMed Scopus Google Scholar, for DNA cleavage by restriction endonuclease Mol. Biol. PubMed Scopus Google Scholar). The SgrAI filament is a with turn D. C.K. regulation of DNA cleavage and through 21: PubMed Scopus Google Scholar). of in the filamentous and a in the of of the to the that be as an an to the of the DNA D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar). of the at the how conformational changes in the protein this which to the active site and a of SgrAI to the DNA The conformational changes first observed in a of SgrAI in the filamentous form to a site DNA, which the phosphate at the cleavage site D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar). However, in this structure, only a Mg2+ observed in the active site in a known as site only Mg2+ observed in structures of SgrAI to DNA M. D. of to of an PubMed Scopus Google Scholar, Horton N.C. in the activation of DNA cleavage by structures of to cleaved DNA and Biol. PubMed Scopus Google Scholar). The in to observed within the filamentous structures a mechanism for activation of the DNA cleavage activity a second Mg2+ binding site is Mg2+ in A are through the which is to be by divalent as as enzymes for the activity of DNA a two PubMed Google Scholar, N.C. DNA The of Google Scholar, A mechanism for PubMed Google Scholar, N.C. the of Struct. Biol. PubMed Scopus Google Scholar, mechanism of on structures. Mol. Biol. PubMed Scopus Google Scholar). In this the two divalent from the the DNA, and the and as the the and the A mechanism for PubMed Google Scholar, N.C. the of Struct. Biol. PubMed Scopus Google Scholar, M. and catalysis and substrate Cell. PubMed Scopus Google Scholar, of structure, and Rev. PubMed Scopus Google Scholar). In SgrAI only site A is The of site by Mg2+ is to be the the DNA cleavage activity of SgrAI in the M. D. of to of an PubMed Scopus Google Scholar, Horton N.C. in the activation of DNA cleavage by structures of to cleaved DNA and Biol. PubMed Scopus Google Scholar). the of two Mg2+ in filamentous SgrAI has been of a site in SgrAI to DNA first observed in the of filamentous SgrAI to a site DNA and D. Horton N.C. and structures of the filament-Forming enzyme mechanisms of activation and substrate Biol. 2022; Scopus Google Scholar). However, is the biologically relevant cofactor for cleavage. The divalent cation is to stall DNA cleavage in an to the active site prior to the DNA cleavage reaction, Mg2+ is to DNA by restriction endonuclease induced by PubMed Google Scholar, Horton N.C. binding in the active site of for the PubMed Google Scholar, activation of enzymes in Rev. PubMed Google Scholar, and PubMed Scopus Google Scholar). enzymatic the enzymatic from the enzymatic the for the between the two has been the of M. and catalysis and substrate Cell. PubMed Scopus Google Scholar, of structure, and Rev. PubMed Scopus Google Scholar, and PubMed Scopus Google Scholar). has a than Mg2+ and is with to the of its and studies of in and Google Scholar, The of relevant to Biol. PubMed Scopus Google Scholar). of to a than Mg2+ of is that of Mg2+ is of in Scholar, and a of Scholar). have been suggested to be the of of DNA cleavage in enzymes by the of insights relevant to enzymes containing Mg2+ binding to a understanding of the enzyme it is to structures with the Mg2+ we present two new structures of filamentous SgrAI containing the biologically relevant cofactor The first structure, resolved to 3.3 Å, the active site mutation which DNA cleavage and an at the cleavage site known as the phosphate The second structure, resolved to 3.1 Å, is from WT SgrAI to an site cleaved DNA in the active site of the cleavage reaction. Both structures the of Mg2+ in A and a model for activation of the DNA cleavage in filamentous which involves the of a binding site for a second Mg2+ at site on these we an mechanistic model for activity of To a of enzymatic it is to stall the enzyme in a To determine the enzymatic configuration of SgrAI and the of within the active site at of we to two of SgrAI containing the Mg2+ The first the mutation which is to enzymatic on the of the Mg2+ in the catalysis. The second the WT which to DNA to and the Both assemblies are to be for structural To the filamentous we SgrAI with a DNA containing the sequence in containing and for 30 at prior to observed filaments on and a containing of the using D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar, D. Horton N.C. and structures of the filament-Forming enzyme mechanisms of activation and substrate Biol. 2022; Scopus Google a resolved to 3.3 Å, which for of Mg2+ a model as a D. Horton N.C. and structures of the filament-Forming enzyme mechanisms of activation and substrate Biol. 2022; Scopus Google to and an atomic model of the filamentous form of SgrAI protein to DNA, a with and a between and model of To the filamentous we WT SgrAI with the same DNA and the to for that the cleavage to the of and we a containing of the a resolved to 3.1 The model and with a between the and model of and the the and atomic for each of the two new structures to each and to structures of SgrAI to The of that the in the form of the protein is to model Mg2+ in the active in A and and the of the is In the of as that the is cleaved in the active site Mg2+ are in the active at the for A and of the mechanism and A of the active site that the of the Mg2+ in A and are are in which be to the of the or and the or of the and be in A and these two structures and of the cleavage mechanism in the of the biologically relevant cofactor site and Mg2+ in and the active site of between Mg2+ and are to as in for shown in To into the atomic of the enzymatic active site prior to DNA we the filamentous to structures of filamentous SgrAI of the active of with the filamentous SgrAI and The in these the divalent in A and are by and Å, The of the of the in A and in is to all with the of it to In the of the is in all with the of and The of the in to the in the active in site The of the in for of the to each with the site A and result in a configuration that is for catalysis with the of DNA cleavage with in of of to structures of WT SgrAI to site DNA with or Mg2+ and cleaved DNA reveals a and of the and with of to However, are observed in the of the the cleavage site and The of site A are and in and the of the site are and that contain a site To into the atomic of the enzymatic active site in the DNA we the filamentous to the structures. to this is to prior filamentous structures. is the of SgrAI in filamentous and forms by changes as a result of the between the two of the SgrAI D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar). The is with the of filamentous SgrAI to Mg2+ and a site DNA in which the is shown in However, we the is in than the the of the of of the cleavage is by Å, and the Mg2+ in site A is by between the two structures the new to that of filamentous SgrAI to and a site DNA containing an The between the two structures is the type of which in or the DNA cleavage. The between and is shown in we that the between the divalent in A are and in site are with the in the of the we a of the as that the structures two of the enzymatic the of to SgrAI which are to changes. a of with the the form to a site DNA and M. D. of to of an PubMed Scopus Google Scholar). this form of the enzyme is with the DNA is site A is in and the between the in site A of and the Mg2+ in site A of is The that the of the than in with of the filamentous The in the within the two structures be to the of DNA cleavage. the of SgrAI containing a cleaved site DNA and Mg2+ Horton N.C. in the activation of DNA cleavage by structures of to cleaved DNA and Biol. PubMed Scopus Google the same as in the In this we in the of the DNA, the and the The of the in the two structures are and the are The in the of the divalent in A and are and Å, to the Mg2+ in in these two structures. In the structure, each Mg2+ to a of the cleaved phosphate In of the to Mg2+ in A and a second site the site Mg2+ to two of the cleaved in only in The of the is the of the in to be to the site A the is to the site Mg2+ in In the structure, the of the cleavage is to the Mg2+ at site this is in In of the two new structures to in the and of the of the cleavage site the the of the in the of the site A and and in the of the the active are relevant to understanding the enzymatic cleavage as be we the two new and A and The assemblies and the structures only by the or of the of in which is of the active site SgrAI to the of with the of two as as a that is a Google Scholar, M. restriction and Mol. PubMed Scopus Google Scholar, and of type restriction PubMed Scopus Google Scholar, restriction and PubMed Scopus Google Scholar). The to the divalent which are for the DNA cleavage reaction, the role of the has been and be The structures with the changes at the which is cleaved in the WT structure, in the The are in the to the of the in the with cleaved for the to between the cleaved phosphate and the DNA cleavage A and are observed in the of the A and The of the site A cation is in the two structures A and the site cation is by between the be by the of the cleaved phosphate from the site binding site and the site A binding which is A and In with the of the the of the active site in these two new structures are all structural and the structural of the filamentous and DNA cleavage. To the binding of the biologically relevant as as the mechanism of activation of we two new structures of filamentous SgrAI to site DNA and Prior structures of filamentous SgrAI using that the DNA cleavage and the active site prior to as or DNA D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar, D. Horton N.C. and structures of the filament-Forming enzyme mechanisms of activation and substrate Biol. 2022; Scopus Google Scholar). these prior structures insights into the conformational changes induced by which the mechanism of DNA cleavage and the structural that from and to stall enzymatic cleavage. in of Mg2+ D. Horton N.C. and structures of the filament-Forming enzyme mechanisms of activation and substrate Biol. 2022; Scopus Google Scholar). Mg2+ and and to in than in the DNA cleavage DNA by restriction endonuclease induced by PubMed Google Scholar, Horton N.C. binding in the active site of for the PubMed Google Scholar). The of this been the of and be to the of to in its or to its to of structure, and Rev. PubMed Scopus Google Scholar, of in Scholar, and a of Scholar). the containing the DNA at the site of DNA and that define the binding configuration of this D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar). The of the of the active as of site by Mg2+ D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar). of the two new structures or and reveals the active site configuration of the WT enzyme with Mg2+ cleavage. The new Mg2+ the mutation in the active site of SgrAI to stall DNA cleavage. Both structures the binding of Mg2+ in A and and the configuration of the DNA, new to our understanding of the mechanism of DNA cleavage by filamentous SgrAI is a divalent DNA and to DNA the two divalent in a cleavage mechanism mechanism first for mechanism of on structures. Mol. Biol. PubMed Scopus Google and the activity of DNA from for the activity of DNA a two PubMed Google has been with to the mechanisms of divalent cation and enzymes N.C. DNA The of Google Scholar, A mechanism for PubMed Google Scholar, N.C. the of Struct. Biol. PubMed Scopus Google Scholar, of structure, and Rev. PubMed Scopus Google Scholar, A mechanism for all PubMed Scopus Google Scholar, for a mechanism of PubMed Scopus Google Scholar, of a PubMed Scopus Google Scholar, D. understanding mechanism and Rev. PubMed Scopus Google Scholar). In this the two divalent are by enzyme as or and at of the to be cleaved The is to an type with a or a mechanism and and the is a D. understanding mechanism and Rev. PubMed Scopus Google Scholar, of cleavage of R. Scholar). The cleavage is by the enzyme of activation of the of the and of the D. understanding mechanism and Rev. PubMed Scopus Google Scholar). are in by the divalent cation in site which a for on the of the The is within from the phosphate and with the that the between the phosphate and the is of the of by DNA from PubMed Google Scholar, of by from PubMed Google Scholar, A new for the of DNA cleavage to the and restriction and to the PubMed Scopus Google Scholar). In to to the site A the of the that it is to a and form the of in Scholar, and a of Scholar). Both A and are to the which an the role of direct or by a D. understanding mechanism and Rev. PubMed Scopus Google Scholar). the which forms on the in the and is to be to of this reaction, as this is and a be D. understanding mechanism and Rev. PubMed Scopus Google Scholar). Many restriction as as containing the restriction endonuclease contain a in active site M. restriction and Mol. PubMed Scopus Google Scholar). of this in of DNA cleavage and DNA binding of restriction endonuclease Biol. PubMed Scopus Google Scholar, M. to in the of the restriction endonuclease 31: PubMed Google Scholar, N.C. DNA cleavage by two in binding PubMed Scopus Google Scholar, N.C. to site specific DNA cleavage by PubMed Scopus Google Scholar). with these our of SgrAI containing the mutation of DNA the of the biologically relevant and Mg2+ it is that the is to the DNA cleavage activity of SgrAI and the role it in cleavage catalysis its the active site has been to as a of activation of the and of the M. restriction and Mol. PubMed Scopus Google Scholar, N.C. DNA cleavage by two in binding PubMed Scopus Google Scholar, N.C. catalysis in type restriction PubMed Scopus Google Scholar, at Biol. Scholar, J.M. activation in an and PubMed Scopus Google Scholar). to these However, a role as a it the of the to be its to our structures that the contacts with the an to its the of the to the and to the site A cation in the active of structures. The of SgrAI is by the of SgrAI to DNA and as as the new of filamentous The cleavage of SgrAI is by the of filamentous Prior to DNA the is to the site A cation and is within a to the of In the cleavage the by the in is by a of the cleaved that this is from the The of the within with the and the of the in the and for activation of the and of the the of the in a in the of the the by the and in in the with a role of the in the are at the of the structure, and the of the be However, activation be by of the by the in of the of the by the J.M. activation in an and PubMed Scopus Google Scholar). this is to the Mg2+ at site a Mg2+ has a of and a of to the of the be J.M. activation in an and PubMed Scopus Google Scholar). In to and the to The is to contain an which is the of the by that is observed in multiple structures to its and increasing the of reaction. SgrAI is of a of enzymes known to form polymeric In the of filamentation in accelerated DNA cleavage and expanded DNA sequence specificity. first filamentation by SgrAI in C.K. Horton N.C. of DNA cleavage by of PubMed Scopus Google Scholar). that we have structures of SgrAI in filamentous and to the structural of the observed activation and of specificity D. C.K. regulation of DNA cleavage and through 21: PubMed Scopus Google Scholar, D. Horton N.C. of activation of the 2019; PubMed Scopus Google Scholar, M. D. of to of an PubMed Scopus Google Scholar, Horton N.C. in the activation of DNA cleavage by structures of to cleaved DNA and Biol. PubMed Scopus Google Scholar, D. Horton N.C. and structures of the filament-Forming enzyme mechanisms of activation and substrate Biol. 2022; Scopus Google Scholar). The two new structures the DNA cleavage mechanism by we for the first Mg2+ site in a filamentous structure, and we the of the which are with the In the prior of filamentous SgrAI with site by an of the and D. Horton N.C. and structures of the filament-Forming enzyme mechanisms of activation and substrate Biol. 2022; Scopus Google which to the of to DNA cleavage by the of the two new filamentous SgrAI using all of a of are in these and of the active with the of the and these structures a of the our model of the DNA cleavage mechanism by a of the with a of a of the Mg2+ in site A In the filamentous the containing and its to a which the binding of a Mg2+ in site of the of the site Mg2+ a of and a of the The site A Mg2+ a that have its for at of in Scholar, and a of Scholar). The is and is for on the the has and on a of the of by DNA from PubMed Google Scholar, of by from PubMed Google Scholar, A new for the of DNA cleavage to the and restriction and to the PubMed Scopus Google Scholar). be a or a D. understanding mechanism and Rev. PubMed Scopus Google Scholar). In our model, the site Mg2+ to the at this and the a in the that forms in the and that be of the The which the of in the of to the of site is to a to the which this two in the SgrAI structures and the we direct by to the and of the prior to and these are observed in the between the site and the is Å, and between the site and the is 3.1 In the structure, the has the site A to to the the is to the site However, direct of the be and from a site be to the to D. understanding mechanism and Rev. PubMed Scopus Google Scholar, J.C. mechanism of DNA cleavage by the restriction enzyme a PubMed Scopus Google Scholar). of the as the from second to with its observed to site in the In the between the of and the Mg2+ in A and in the are long for direct and Å, be a of in or to the observed in the of the we that the site A cation to in our structural the site cation on to and to during the The and of enzyme filamentation are only that is is that in the filament over as to the enzyme in an or an as in the of SgrAI C.K. Horton N.C. Novel insights into filament-Forming enzymes.Nat. Rev. Mol. Cell Biol. 2020; 21: 1-2Crossref PubMed Scopus (11) Google Scholar, D. Horton N.C. The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease Sgrai.Biochem. Soc. Trans. 2022; 50: 1703-1714Crossref PubMed Scopus (0) Google Scholar, 7Hvorecny K.L. Kollman J.M. Greater than the Sum of parts: mechanisms of metabolic regulation by enzyme filaments.Curr. Opin. Struct. Biol. 2023; 79102530Crossref PubMed Scopus (11) Google Scholar). filamentation to enzymes as during of E.M. J.M. for of Scopus Google and to as a to a of active enzymes in the E.M. in the activity in 2020; PubMed Scopus Google Scholar). filamentation substrate binding the of an enzyme for its K.L. Kollman J.M. Greater than the Sum of parts: mechanisms of metabolic regulation by enzyme filaments.Curr. Opin. Struct. Biol. 2023; 79102530Crossref PubMed Scopus (11) Google Scholar, A.L. M. J.C. D. filament to Struct. Mol. Biol. 2022; PubMed Scopus Google Scholar, E.M. Kollman J.M. structural regulation of Struct. Mol. Biol. 2020; PubMed Scopus

  PublisherOA PDFDOI

• Two-Metal Ion Mechanism of DNA Cleavage by Activated, Filamentous SgrAI

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-05-01

  preprintOpen accessSenior authorCorresponding

  Abstract Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments. SgrAI filamentation increases enzymatic activity by up to three orders of magnitude and additionally expands its DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-electron microscopy (cryo-EM). The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg 2+ and define how the Mg 2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg 2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.

  PublisherOA PDFDOI

• DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease

  Biochemistry · 2024-01-11 · 2 citations

  articleSenior authorCorresponding

  Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

  PublisherDOI

• Data for PNCC Project 160207 from January 2023

  OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-01

  datasetOpen access1st authorCorresponding
  PublisherDOI

• Data for PNCC Project 160207 from January 2023

  OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-01

  datasetOpen access1st authorCorresponding
  PublisherDOI

• Data for PNCC Project 160207 from January 2023

  OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-01

  datasetOpen access1st authorCorresponding
  PublisherDOI

• Data for PNCC Project 160207 from January 2023

  OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-01

  datasetOpen access1st authorCorresponding
  PublisherDOI

• Data for PNCC Project 160207 from January 2023

  OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-01

  datasetOpen access1st authorCorresponding
  PublisherDOI

Recent grants

• NIH Grant R01GM066805

  NIH · $1.2M · 2010

• Collaborative Research: Structures, Mechanism, and Functional Relevance of Filament Formation by Non-Cytoskeletal Enzymes

  NSF · $1.1M · 2019–2025

• NIH Grant P41RR001209

  NIH · $12.9M · 2015

• Protein Filament Formation in Activating and Modulating Enzymatic DNA Cleavage Specificity

  NSF · $762k · 2014–2020

Frequent coauthors

• Noura Darwish

  Nestlé (Switzerland)

  34 shared

• Chad K. Park

  University of Arizona

  20 shared

• Dmitry Lyumkis

  Salk Institute for Biological Studies

  17 shared

• John J. Perona

  Portland State University

  12 shared

• Niloofar Ghadirian

  University of Arizona

  10 shared

• Pete Dunten

  Stanford Synchrotron Radiation Lightsource

  9 shared

• Jonathan L. Sanchez

  George Mason University

  9 shared

• Jurate Bitinaite

  New England Biolabs (United States)

  8 shared

Labs

• Horton LabPI

Education

• Ph.D., Department of Chemistry

  Univeristy of Pennsylvania

  1994

• B.S., Chemistry

  Southern Illinois University Carbondale

  1986