个人简介

Contributing member in DNA Replication and Repair, Faculty of 1000, 2010-pres Nielsen Trust Fund Award, College of Medicine, 2001 NSF POWRE Award, 1999-2001 American Cancer Society Postdoctoral Fellow, 1997-1999

研究领域

Biochemistry/Nucleic Acids and Genomes/Protein and Membrane Biochemistry/Structural Biology

Control of enzyme activity by enzyme polymerization Phage-host systems are under intense evolutionary pressure, consequently they have developed remarkably ingenious mechanisms of attack and defense. Our lab investigates one such remarkable system: that found in Streptomyces griseus. Based on its biochemical activities, SgrAI, a nucelase from S. griseus, is postulated to be activated by binding to invading phage DNA, simultaneously expanding its DNA sequence cleavage specificity and forming polymers that may act to protect the host DNA from its resulting off-target cleavage activity. Enzyme mechanisms involving polymer or filament formation are exceedingly rare, although recent screens suggest this may be more common than previously thought. Being a potentially new paradigm for enzyme regulation, several fundamental questions arise that are under investigation in our lab, including the structure, kinetics, and biological role of the polymer. Our previous biochemical data suggests that the polymer formed from activated SgrAI is a run-on oligomer, which has now been confirmed by the 8.6 Å cryo-electron microscopy structure determined in our lab. Although this structure shows how the SgrAI dimers bound to activating DNA associate in a repeating helical arrangement, fundamental questions such as how DNA cleavage is activated, how DNA sequence specificity is altered, and whether or not domain swapping (found in a crystal structure of two DNA bound SgrAI dimers) is present require higher resolution and therefore remain to be answered. Also important to understanding the function of the run-on oligomer is determining how formation of such an assembly, where the bound DNA appears critical for oligomer stability, accelerates rather than impedes multiple DNA cleavages. Finally, the biological role for run-on oligomer formation has been hypothesized to function in protecting the host DNA from dangerous off-target cleavages made possible via activation of SgrAI, by sequestering SgrAI on the invading phage DNA. Current studies in our lab aim to investigate the structure of the run-on oligomer using biochemical and x-ray crystallographic methods, measure kinetic steps involving polymer formation and dissociation in the reaction pathway using pre-steady state fluorescence methods, and test the postulated biological role of the polymer using in vitro and in vivo assays including phage infection challenges.

近期论文

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Lyumikis D., Talley, H., Stewart, A., Shah, S., Park, C.K., Tama, F., Potter, C.S., Carragher, B., Horton, N.C. (2013) "Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization", Structure, 21, 1848-1858. Ma, X., Shah, S., Zhou, M., Park, C.K., Wysocki, V.H., Horton, N.C. (2013) "Structural Analysis of Activated SgrAI-DNA Oligomers Using Ion Mobility Mass Spectrometry", Biochemistry, 52, 4373-81. Little, E.J., Dunten, P.W., Bitinaite, J. & Horton, N.C. (2011) "New Clues in the Allosteric Activation of DNA Cleavage by SgrAI; Structures of SgrAI Bound to Cleaved Primary Site DNA and Uncleaved Secondary Site DNA", Acta Cryst., 67, 67-74. Park, C.K., Stiteler, A.P., Shah, S., Ghare, M.I., Bitinaite, J. & Horton, N.C. (2010) "Activation of DNA Cleavage by Oligomerization of DNA bound SgrAI", Biochemistry, 49, 8818-8830. Horton, N.C. & Park, C.K., (2010) "Crystallization of Zinc Finger Proteins bound to DNA", Methods Mol Biol. 649:457-77. Park, C.K., Joshi, H.K., Agrawal, A., Ghare, M.I., Little, E.J., Dunten, P.W., Bitinaite, J. & Horton, N.C. (2010) "Domain Swapping in Allosteric Modulation of DNA Specificity", PLoS Biology, 8(12):e1000554. Dunten, P.W., Little, E.J. & Horton, N.C. (2009), "The restriction enzyme SgrAI: structure solution via combination of poor MIRAS and MR phases.", Acta Cryst. D Biol Crystallogr., 65, 393-8. Little, E.J., Babic, A.C., & Horton, N.C. (2008) "Early interrogation and recognition of DNA sequence by indirect readout", Structure 16, 1828-37. (Given F1000 "must read") Babic, A.C., Little, E.J., Manohar, V.M., Bitinaite, J., & Horton, N.C. (2008) "DNA distortion and specificity in a sequence-specific endonuclease" J. Mol. Biol. 383, 186-204. Dunten, P.W., Little, E.J., Gregory, M.T., Manohar, V.M., Dalton, M., Hough, D., Bitinaite, J., Horton, N.C. (2008) "The structure of SgrAI bound to DNA; recognition of an 8 base pair target", Nucleic Acids Res. 36, 5405-16. Horton, N.C. (2007) "Deoxyribonucleases". In Protein-Nucleic Acid Interactions: Structural Biology; Carl C. Correll, Pheobe Rice, Eds.; RSC Publishing: Cambridge, United Kingdom, 2008. Segal, D.J., Crotty, J., Bhakta, M., Barbas III, C.F. & Horton, N.C. (2006) "Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA", J. Mol. Biol. 363, 405-421. Joshi, H.K., Etzkorn, C., Chatwell, L., Bitinaite, J., & Horton, N.C. (2006) "Alteration of sequence specificity of the type II restriction endonuclease HincII through an indirect readout mechanism." J. Biol. Chem. 281, 23852-69. Little, E.J. & Horton, N.C. (2005) "DNA induced conformational changes in type II endonucleases; the structure of unliganded HincII" J. Mol. Biol. 351, 76-88.