NGEC Principal Investigator
Member, Division of Basic Sciences
Fred Hutchinson Cancer Research Center
Division of Basic Sciences
Program in Structural Biology
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N.
Seattle, WA 98109
Tel: (206) 667-4031 Fax: (206) 667-3331
E-mail Dr. Stoddard
Dr. Stoddard graduated from Whitman College with a BA in chemistry, and earned his PhD in biophysical chemistry from the Massachusetts Institute of Technology (MIT). He was a Whitaker College Health Sciences Graduate Research Fellow at MIT, and a Helen Hay Whitney Postdoctoral Research Fellow at University of California, Berkeley before joining Fred Hutchinson Cancer Research Center (FHCRC) as an assistant member. Now a full member at FHCRC, Dr. Stoddard is also an affiliate faculty in the Department of Biochemistry at the University of Washington School of Medicine and a member of the Faculty of 1000 (Biocatalysis Division).
Dr. Stoddard is the principal investigator for the NGEC in the area of Structural and Biophysical Studies of Novel Homing Endonucleases.
“The NGEC and its collaborative interactions offer an unparalleled opportunity to translate structural biology and protein engineering into significant new therapeutic strategies and studies.”Homing endonucleases are extraordinarily specific DNA-binding proteins, acting specifically at individual sites within a host genome. These proteins are under intense study for the purpose of engineering single chain gene-specific reagents to be used for gene therapy and other applications.
Over the past 10 years, the Stoddard laboratory has determined the structure and mechanisms of representatives form all known families of homing endonucleases, found respectively in phage, eubacteria, archae, and single cell eukarya. In addition, the Stoddard laboratory has described the creation of homing endonuclease variants that act at noncognate sites. These constructs have been generated using both bacterial selection strategies and computational methods, both of which target enzyme residues that directly contact DNA base pairs. In either case, such experiments have produced endonucleases that display shifted DNA recognition properties, but at the cost of reduced site-discrimination abilities.
Dr. Stoddard hypothesizes that in order to completely reprogram the DNA recognition specificity of a homing endonuclease, without a reduction in site discrimination, the re-sculpting of protein-DNA contacts must be combined with the selection of structural mutations in the nearby enzyme scaffold that "fine-tune" the protein -DNA interaface of each novel cognate complex. The goal of overall Specific Aim #1 of the NGEC is to accomplish this task by combining random mutation of the endonuclease scaffold, computational redesign and selection of DNA contacts, and biochemical/biophysical characterization of the resulting endonuclease constructs.
The Stoddard laboratory will be responsible for the following aims in its component of the NGEC activities:
As part of its role in the NGEC, the Stoddard laboratory is seeking to answer questions such as: What is the structural basis for the DNA binding specificity and catalytic activity of homing endonucleases generated by the Scharenberg, Monnat and Baker laboratories, within the NGEC? How do these properties compare to those of naturally occurring (i.e. wild-type) homing endonucleases?
Key lab personnel assisting this work include: Amanda Mak, PhD (postdoctoral fellow); Lei Zhao, BS (graduate research fellow); and Ryo Takeuchi, PhD (postdoctoral fellow).
Abbie Lambert, graduate student, and Ryo Takeuchi, post doctoral fellow, from Barry Stoddard’s lab.
The Stoddard lab focuses its research on the structure, function and mechanism of rare-cutting endonucleases, particularly intron-encoded homing endonucleases.
The overall goal of the Stoddard lab is to characterize the structure/function relationships of a variety of enzymatic catalysts at the atomic level. Much of this work is being extended into efforts to engineer novel properties onto existing enzyme scaffolds. Most of the lab’s particular area of focus is on enzymes that act to modify nucleic acid substrates, and a unifying theme between many of the individual projects is the selection and engineering of these enzymes for targeted therapeutic applications.
The Stoddard lab uses cutting-edge technology and other tools, including macromolecular X-ray crystallography, computer modeling, calorimetric measurements of binding thermodynamics, and genetic manipulation of the molecules of interest, combined with biochemical analyses of enzyme function.
Over the past 10 years, Dr. Stoddard and his research team have determined the structure and mechanism of representatives of each of the known families of homing endonucleases. In collaboration with the Monnat and Baker laboratories, the Stoddard lab has also described a series of studies in which the structure of several of these proteins was engineered in order to alter their DNA specificity. These latter studies have paved the way for eventual development and application of homing endonucleases as gene specific reagents.
Stephens, K., Monnat, R. Heath, P.J. and Stoddard, B. L. (1997) "Crystallization and preliminary x-ray characterization of I-CreI: an intron-encoded restriction endonuclease" Proteins 28: 137 - 139.
Heath, P. J., Stephens, K., Monnat, R. and Stoddard, B. L. (1997) "Structure of the I-CreI intron-encoded endonuclease: a novel fold that binds and cleaves a long DNA target sequence" Nature Structural Biology 4 (6) 468-476.
Flick, K.E., McHugh, D., Heath, J.D., Stephens, K.M., Monnat, R.J. Jr., and Stoddard, B.L. (1997) "Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum" Protein Science 6: 2677 - 2680.
Flick, K.E., Jurica, M. S., Monnat, R. J. Jr., and Stoddard, B. L. (1998) "DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI" Nature 394: 96 - 101.
Jurica, M. S., Monnat, R. J. Jr. and Stoddard, B. L. (1998) "DNA recognition and cleavage by the LAGLIDADG homing endonuclease I-CreI" Molecular Cell 2: 469 - 476.
Galburt, E., Chevalier, B., Tang, W., Jurica, M. S., Flick, K. E., Monnat, R. J. Jr., and Stoddard, B. L. (1999) "A novel endonuclease mechanism directly visualized for I-PpoI" Nature Structural Biology 6: 1096 - 1099.
Galburt, E., Jurica, M., Chevalier, B., Erho, D., Tang, W., Monnat, R. J. Jr., and Stoddard, B. L. (2000) "Conformational changes and cleavage by the homing endonuclease I-PpoI: a critical role for a leucine residue in the active site" J. Mol. Biol. 300(4): 877 - 887.
Chevalier, B., Monnat, R. J. Jr. and Stoddard, B. L. (2001) "The homing endonuclease I-CreI uses three metals, one of which is shared between two active sites" Nature Structural Biology 8 (4): 312 - 316.
Chevalier, B., Kortemme, T., Chadsey, M., Baker, D., Monnat, R. J. Jr. and Stoddard, B. L. (2002) "Design, activity and structure of a highly specific artificial endonuclease" Molecular Cell 10 : 895 - 905. (Also listed under "protein engineering" below)
Chevalier, B., Turmel, M., Lemieux, C., Monnat, R. J. Jr. and Stoddard, B. L. (2003) "Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI" J. Mol. Biol. 329: 253 - 269.
Bolduc, J. M., Spiegel, P. C., Chatterjee, P., Brady, K. L., Downing, M. EW., Caprara, M. G., Waring, R. B. and Stoddard, B. L. (2003) "Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor" Genes and Development 17: 2875-2888. (See also the corresponding commentary by Marlene Belfort)
Bakhrat, A., Jurica, M. S., Stoddard, B. L. and Raveh, D. (2004) "Homology modeling and mutational analysis of HO endonuclease of yeast" Genetics 166: 721 - 728.
Shen, B. W., Landthaler, M., Shub, D. A. and Stoddard, B. L. (2004) "DNA binding and cleavage by the HNH homing endonuclease I-HmuI" J. Mol. Biol. 342 (1): 34 - 56..
Sussman, D., Chadsey, M., Fauce, S., Engel, A., Bruett, A., Monnat, R. J. Jr., Stoddard, B. L. and Seligman, L. (2004) "Isolation and characterization of new homing endonuclease specificities at individual target site positions" J. Mol. Biol. 342 (1): 31 - 41.
Chevalier, B., Sussman, D., Otis, C., Noel, A. -J., Turmel, M., Lemieux, C., Stephens, K., Monnat, R. J. Jr. and Stoddard, B. L. (2004) "Analysis of the symmetric metal-dependent I-CreI homing endonuclease mechanism" Biochemistry 43: 14015 - 14026.
Landthaler, M., Shen, B.W., Stoddard, B. L. and Shub, D.A. (2006) "Two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities" J. Mol. Biol. 358 (4): 1137 - 1151.
Ashworth, J., Havranek, J. J., Duarte, C. M., Sussman, D., Monnat, R. J. Jr., Stoddard, B. L. and Baker, D. (2006) "Computational redesign of endonuclease DNA binding and cleavage specificity" Nature Nature 441: 656 - 659.. (Also listed under "protein engineering" below) (Also see this news and views by Carl Pabo)
Spiegel, P. C., Chevalier, B., Sussman, D., Turmel, M., Lemieux, and Stoddard, B. L. (2006) "The structure of I-CeuI homing endonuclease: evolving asymmetric DNA recognition from a symmetric protein scaffold" Structure 14 (5): 869 - 880. (Also see this commentary by Fred Gimble)
Rosen, L. E., Morrison, H. A., Masri, S., Brown, M. J., Springstubb, B., Sussman, D., Stoddard, B. L. and Seligman, L. M. (2006) "Homing endonuclease I-CreI derivatives with novel DNA target specificities" Nuc. Acids Research 34 (17): 4791 - 800.
Zhao, L, Boncora, R., Shub, D. and Stoddard, B. L. “The restriction folds turns to the dark side: a bacterial homing endonuclease with a PD(D/E)XK motif” (2007) EMBO Journal 26: 2432 - 2442. (Zhao et al. supplementary information)
Volna, P., Jarjour, J., Baxter,S., Roffler, S. R., Monnat, R. J. Jr., Stoddard, B. L. and Scharenberg, A. M. (2007) "Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases" Nucleic Acids Research 35 (8): 2748 - 2758. (Volna et al. supplementary information)
Eastberg, J., Monnat, R. J. Jr., and Stoddard, B. L. (2007) “Mutability of the ßßa-metal HNH nuclease active site: substitution of a deprotonation mechanism” Biochemistry 46 (24): 7215 - 7225.
Scalley-Kim, M., McConnell-Smith, A. and Stoddard, B. L. (2007) "Coevolution of homing endonuclease specificity and its host target sequence" J. Mol. Biol. 372 (5): 1305 - 1319. (plus Supplementary Information).
Eastberg, J. H., McConnell-Smith, A., Zhao, L., Ashworth, J., Shen, B. W. and Stoddard, B. L. (2007) "The thermodynamics of DNA target site recognition by homing endonucleases" submitted.
Eastberg, J., Monnat, R. J. Jr., and Stoddard, B. L. (2007) “Mutability of the ßßa-metal HNH nuclease active site: substitution of a deprotonation mechanism” Biochemistry 46 (24): 7215 - 7225.
Scalley-Kim, M., McConnell-Smith, A. and Stoddard, B. L. (2007) "Coevolution of homing endonuclease specificity and its host target sequence" J. Mol. Biol. 372 (5): 1305 - 1319. (Plus supplementary Information)