Barry Stoddard, PhD
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
Dr. Stoddard is a full member of the Fred Hutchinson Cancer Research Center. His research interests include understanding structure/function relationships of biological systems at the atomic level, employing X-ray crystallography, computer modeling, and genetic manipulation of the molecules of interest, and he has been a pioneer in applying these tools to the structural and biochemical analysis of various types of homing endonuclease proteins. His laboratory is presently focusing on two problems which share relevance to human disease and genetically targeted therapeutic applications - engineering of novel homing endonuclease variants for targeted genome modification, and engineering of nucleotide synthesis and salvage enzymes for directed anti-cancer therapy.
Dr. Stoddard is the principal investigator for the NGEC in the area of structural and biophysical studies of novel homing endonucleases.
Awards and Honors
- AAAS Newcomb Cleveland Prize: 2005
Areas of Expertise
- Macromolecular X-ray crystallography
- Structural enzymology
- Structural biology
- Enzyme catalysis
- Protein design
- DNA metabolism and modification
Overview of the Stoddard 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.
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 resculpting of protein-DNA contacts must be combined with the selection of structural mutations in the nearby enzyme scaffold that "fine-tune" the protein-DNA interface 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:
- It will determine the DNA recognition specificity profile of the novel endonuclease constructs using two related methods to directly visualize cleavage of DNA target variants and to quantitate specificity at each base pair.
- It will determine the thermodynamic signature of cognate and non-cognate site recognition for redesigned homing endonucleases, using isothermal titration calorimetry (ITC).
- It will determine the three-dimensional structure of novel endonuclease-DNA cognate pairs at high resolution, and will characterize (a) the effect of enzyme scaffold mutations on backbone structure, and (b) the accuracy of computational redesign predictions within the protein-DNA interface.
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 personnel carrying out this research include Ryo Takeuchi (postdoctoral fellow), Amanda Mak (postdoctoral fellow), Michael Metzger (postdoctroal fellow) and Greg Taylor (graduate student).