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, 1989-1995; 2004-2007
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, University of Toronto).
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 [M. M. Skinner et al. 1994. Proc. Natl. Acad. Sci., USA 91:2071-2075] and the superhelical structure of the complex with single-stranded DNA has been examined by electron microscopy and X-ray scattering [C. W. Gray. 1989. J. Mol. Biol. 208:57-64; G. A. Olah et al. 1995. J. Mol. Biol. 249:576-594].
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). An unexpected finding was that DNAs selectively saturated by g5p were G-rich and formed G-quadruplex structures, as detected by CD (circular dichroism) spectroscopy and other techniques [J.-D. Wen et al. 2001. Biochemistry 40:9300-9310; J.-D. Wen and D. M. Gray. 2002. Biochemistry 41:11438-11448]. The central 26-nucleotide portion of one proposed G-quadruplex sequence that is initially bound by g5p is shown in the figure below. This sequence is bounded by 16-nucleotide tails at the 3' and 5' ends.
The 3' and 5' tails are subsequently bound by g5p 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 [A. W. Oliver et al. 2000. J. Mol. Biol. 301:575-84], 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.
The Human Replication Protein A
An essential human single-stranded DNA binding protein, replication protein A (hRPA), is capable of melting triplexes and some telomeric quadruplex DNA structures under low salt conditions. We have begun to study the circular dichroism and electrophoretic mobility of hRPA complexes that form with various DNA sequences. We find that hRPA can bind and melt non-telomeric, intramolecular DNA G-quadruplexes, such as shown in the above figure, under physiological conditions (200 mM KCl and 37 °C). The hRPA binds to the 58-mer that contains this embedded quadruplex with an affinity equal to or greater than that to non-quadruplex forming 58-mers selected from the same library of sequences.
D. M. Gray, C. W. Gray, B.-H. Yoo, and T.-F. Lou. 2010. Antisense DNA parameters derived from next-nearest-neighbor analysis of experimental data. BMC Bioinformatics 11:252.
J.-H. Fan, E. Bochkareva, A. Bochkarev, and D. M. Gray. 2009. Circular dichroism spectra and electrophoretic mobility shift assays show that human replication protein A binds and melts intramolecular G-quadruplex structures. Biochemistry 48:1099-1111.
D. M. Gray, J.-D. Wen, C. W. Gray, R. Repges, C. Repges, G. Raabe, and J. Fleischhauer. 2008. Measured and calculated CD spectra of G-quartets stacked with the same or opposite polarities. Chirality 20:431-440.
T.-C. Mou, M. Shen, S. Abdalla, D. DelaMora, E. Bochkareva, A. Bochkarev, and D. M. Gray. 2006. Effects of ssDNA sequences on non-sequence-specific protein binding. Chirality 18:370-382.
J.-D. Wen, 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, pp. 1-10.
B. H. Yoo, 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. Nucl. Acids. Res. 32:2008-2016.
W.-Q. Zou, J. Zheng, D. M. Gray, P. Gambetti, and S. G. Chen. 2004. Antibody to DNA detects scrapie but not normal prion protein. Proc. Natl. Acad. Sci., USA 101:1380-1385.
J. D. Wen, and D. M. Gray. 2004. Ff gene 5 single-stranded DNA-binding protein assembles on nucleotides constrained by a DNA hairpin. Biochemistry 43:2622-2634.
T.-F. Lou, 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.
T.-C. Mou, 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.
J.-D. Wen, and D. M. Gray. 2002. The Ff gene 5 protein single-stranded DNA-binding protein binds to the transiently folded form of an intramolecular G-quadruplex. Biochemistry 41:11438-11448.
A. B. Upadhyaya, M. Khan, T.-C. Mou, M. Junker, D. M. Gray, and J. DeJong. 2002. The germ-cell-specific transcription factor ALF. J. Biol. Chem. 277:34208-34216.
D. M. Gray, C. W. Gray, T.-C. Mou, and J.-D. Wen. 2002. CD of single-stranded, double-stranded, and G-quartet nucleic acids in complexes with a single-stranded DNA-binding protein. Enantiomer 7:49-58.
T.-C. Mou, N. Sreerama, T. C. Terwilliger, R. W. Woody, and D. M. Gray. 2002. Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions. Protein Science 11:601-613.
T.-C. Mou, and D. M. Gray. 2002. The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2'-O-methyl modifications. Nucleic Acids Res. 30:749-758.
J.-D. Wen, C. W. Gray, and D. M. Gray. 2001. SELEX selection of high-affinity oligonucleotides for bacteriophage Ff gene 5 protein. Biochemistry 40:9300-9310.
T.-C. Mou, C. W. Gray, T. C. Terwilliger, and D. M. Gray. 2001. Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA. Biochemistry 40:2267-2275.
D. M. Gray. 2000. CD of protein-nucleic acid interactions, in "Circular Dichroism: Principles and Applications," 2nd Ed. (Eds. N. Berova, K. Nakanishi, and R. W. Woody) John Wiley & Sons, New York, NY pp. 769-796.
- Updated: September 23, 2008