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.
Single particle reconstruction of SgrAI/DNA polymer using cryo-electron microscopy (CryoEM). A. Top and side views showing the helical organization of the SgrAI polymer. Eight distinct DNA binding domains (DBD) are shown in distinct colors. B. Helical reconstruction of the SgrAI/DNA polymer at 8.6 Å resolution using cryoEM data. SgrAI shown as light and dark orange, DNA shown as dark blue. C. CryoEM envelope around one DNA bound SgrAI dimer, shown from three different views. D. As in C with transparency and the ribbon drawing of the fitted coordinates of SgrAI bound to DNA derived from x-ray crystallography. The two subunits of the SgrAI dimer from the polymer undergo a 10° rotation relative to the x-ray crystallographic structure of the free, unpolymerized SgrAI/DNA complex. (Adapted from Lyumkis, et al., 2013, Structure 21, 1848-1858.)
Triggering of Autoimmune Disease by Viral Infection
Human Parvovirus B19 (B19V) is a small single stranded DNA virus that infects the majority of the population. Though most individuals show no lasting effects from the viral infection, several very serious conditions (aplastic crisis, pure red-cell aplasia, and hydrops fetalis) are known to occur in susceptible populations, and many instances of liver, heart, and autoimmune disease following infection have also been reported. Yet, despite the ubiquity of the virus and the rare but serious complications, few detailed molecular studies have been performed with B19V or its components. We propose to begin such studies by investigating the roles of the main B19V replication protein, NS1, and its role in two biological processes, namely viral replication and host gene transactivation. Of interest is how NS1 accomplishes these processes which include specific recognition of DNA and host proteins, as well as manipulation of DNA including cleavage and alterations of DNA structure. These studies will make use of both purified, recombinant NS1 protein, as well as NS1 expressed in different human cell lines, and will build the groundwork for future studies in elucidating the role of NS1 in the different disease states, as well as how interactions with different cellular environments, including different genetic backgrounds, leads to these disease states.
Model of human parvovirus B19 NS1 protein nuclease domains interacting with duplex DNA.
Sanchez, J.L., Romero, Z., Quinones, A., Torgesun, K., and Horton, N.C. (2016) "DNA Binding and Cleavage by the Human Parvovirus B19 NS1 nuclease domain", Biochemistry, in press, PMID: 27809499, DOI: 10.1021/acs.biochem.6b00534.
Shah, S., Sanchez, J., Stewart, A., Piperakis, M.M., Cosstick, R., Nichols, C., Park, C.K., Ma, X., Wysocki, V., Bitinaite, J., Horton, N.C. (2015) “Probing the Run-On Oligomer of Activated SgrAI Bound to DNA”. PLoS One. Apr 16;10(4):e0124783. doi: 10.1371/journal.pone.0124783. eCollection 2015. PubMed PMID: 25880668; PubMed Central PMCID: PMC4399878.
Shah, S, Dunten, P., Stiteler, A., Park, C.K. & Horton, N.C. (2014) “Structure and Specificity of FEN-1 from Methanopyrus kandleri”, Proteins, Oct 30. doi: 10.1002/prot.24704.
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.