Donald Gray, Ph.D.
Education and Professional Affiliations
B.A., Math, Physics, and Chemistry, Susquehanna University
M.S., Molecular Biophysics, Yale University
Ph.D., Molecular Biophysics, Yale University
Member, Biophysical Society
Member, American Association for the Advancement of Science
Member, American Chemical Society
Department Head, 2004-present; 1989-1995
Single-stranded DNA Binding Proteins
Our studies are focused on the bacterial virus Ff gene 5 protein, g5p (in collaboration with Dr. T. Terwilliger, Los Alamos National Laboratory) and the human replication protein A (in collaboration with Dr. A Bochkarev,
The fd Gene 5 Protein
The Ff gene 5 protein is an excellent model single-strand DNA binding protein to study, because the crystallographic structure of the protein dimer is known [1] and the superhelical structure of the complex with single-stranded DNA has been examined by electron microscopy and X-ray scattering [2, 3].
SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been successfully used to isolate, from an ssDNA library of 58-mers, those sequences that bind g5p with high-affinity under physiologically relevant conditions (200 mM NaCl, 37°C, and pH 7.4). Our first unexpected finding was that the selected DNAs were G-rich and formed G-quadruplex structures, as detected by CD (circular dichroism) spectroscopy and other techniques [4, 5]. The portion of one G-quadruplex sequence that is initially bound by g5p dimers to form an initiation complex is shown in Figure 1. The 3' and 5' tails are subsequently bound to form a saturated complex. Although the binding of g5p to the SELEX-selected G-quadruplex differs from that to the G-quadruplex described by Kneale and coworkers [6, 7], in both cases nucleotides are constrained in positions favorable for binding. That is, high affinity sites for g5p binding may consist of appropriately constrained regions of ssDNA to reduce the entropic cost of binding.
In a second set of SELEX experiments another unprecedented finding was that g5p binds to DNA hairpins that have the additional ability to dimerize (Figure 2) [8]. The equilibrium between dimer and monomer DNA hairpin can be shifted by adding the 3' or 5' primers. The finding that g5p assembles on hairpin-constrained nucleotides points to a new dimension of how this small ssDNA-binding protein might achieve its sequence-specific binding functions. Thus, from the study of interactions between the g5p and SELEX-selected sequences, we have found that two distinct types of DNA structures, G-quadruplexes and hairpins, can provide high-affinity sites for g5p binding. Of these two, hairpins may be the more relevant in the biological functioning of the g5p. A search of the fd genome yielded only six G4-5 blocks, two of which are located in or near the gene 2 operator responsible for g5p-mediated translational repression [9]. In contrast, based on mfold calculations, there are more than one hundred hairpins distributed throughout the fd DNA genome. Moreover, mfold calculations with the RNA yield extensive stem-loop structures, with mismatches and bulge loops, that involve the mRNA leader sequences for all five viral genes 1, 2, 3, 5, and 10 that are controlled by g5p. Favorably constrained nucleotides in such complex RNA stem-loop structures may provide excellent targets for g5p binding, although this remains to be tested.
Recent Publications:
1. Mou, T.C., M.C. Shen, T.C. Terwilliger and D.M. Gray. 2003. Binding and Reversible Denaturation of Double-Stranded DNA by Ff Gene 5 Protein. Biopolymers. 70:637-648.
2. Lou, T.F., C.W. Gray and D.M. Gray. 2003. The Reduction of Raf-1 Protein by Phosphorothioate ODNs and siRNAs Targeted to the Same Two mRNA Sequences. Oligonucleotides. 13:313-324.
3. Zou, W.Q., J. Zheng, D.M. Gray, P. Gambetti and S.G. Chen. 2004. Antibody to DNA detects scrapie but not normal prion protein. PNAS. 101:1380-1385.
4. Yoo, B.H., E. Bochkareva, A. Bochkarev, T.C. Mou and D.M. Gray. 2004. 2'-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro. Nucleic Acids Research. 32:2008-2016.
5. Wen, J.D. and D.M. Gray. 2004. Ff Gene 5 Single-Stranded DNA-Binding Protein Assembles on Nucleotides Constrained by a DNA Hairpin. Biochemistry. 9:2622-263.
6. Wen, J.-D. and D.M. Gray. 2004. Selection of Genomic Sequences That Bind Tightly to Ff Gene 5 Protein: Primer-Free Genomic SELEX. Nucl. Acids. Res. 32, e182:1-10.
7. Mou, T.-C., M. Shen, S. Abdalla, D. DelaMora, E. Bochkareva, A. Bochkarev, & D. M. Gray. 2006. Effects of ssDNA Sequences on Non-Sequence-Specific Protein Binding. Chirality 18: 370-382.
- Updated: November 9, 2007
