Jacques R. Fresco
Lewis Thomas Lab, 127
A new approach to gene therapy; Cytogenetic probes for amplified cancer-relevant genes; Physical-Chemical studies of nucleic acids; Mutagenic mechanisms; Molecular mechanisms
One major facet of our research depends on nucleic acid third-strand binding to Watson-Crick duplexes. Work in this laboratory and elsewhere has shown that:
There is a third-strand binding code by which a residue in a third strand can specifically recognize a base pair in a homopurine•homopyrimidine target segment of a complementary Watson-Crick duplex by hydrogen-bonding to it.
Third strands can contain both purine and pyrimidine residues in irregular sequences.
Third-strand orientation relative to the homopurine strand of the target duplex can vary depending upon third-strand base composition.
Third-strand pyrimidine residues only recognize and are hydrogen-bonded in the Hoogsteen orientation to the purine member of a Watson-Crick pair of a target duplex, whereas purine third-strand residues recognize and are hydrogen-bonded to either or both the purine and the pyrimidine members of a target Watson-Crick pair, depending upon third-strand orientation. The resulting hydrogen-bonding schemes provide a rationale for the specificity inherent in the third-strand binding code-that is, why certain base triplets are forbidden.
Our current work is directed toward applying third-strand binding for four purposes:
Gene repair therapy, i.e., repairing mutated genes responsible for inherited diseases. We have selected the hemoglobin mutation responsible for Sickle-Cell Anemia as the demonstration mutation for repair of this gene in hematopoietic stem cells. We have developed a specific third strand that can bind to the target and invade the adjacent duplex sequence. This strand is specifically photo-coupled to the mutant base in the coding strand, which is then repaired during bypass replication of DNA.
In situ hybridization to specific gene or other target sequences in chromosomes for the purpose of identifying and quantitating their copy number. This approach is being particularly applied to the determination of the degree of amplification of genes that become multicopy in various types of cancer, e.g., Her2/neu and topo II in breast cancer. For these purposes we employ short third strands labeled with fluorescent dyes.
Identifying, sorting, and bulk isolation of individual human chromosomes. These various applications exploit the remarkable sequence specificity of third-strand binding to target duplexes.
Binding to gene target sites in vivo to alter their functional roles.
Physical-chemical studies of nucleic acids
We are investigating conditions under which the stability of DNA is directly proportional to the A•T rather than the G•C content.
We are investigating how conditions of macromolecular crowding inside cells enhance the stability of various nucleic acid helical complexes.
We are experimentally evaluating the possible roles of unfavored tautomers as a source of base mispairing events in base pair-dependent reactions of nucleic acid and protein synthesis. Recently, we identified and determined the equilibrium concentration of the unfavored tautomer of the base analog mutagen 5-hydroxy-deoxyCytidine, for which we showed by UV resonance Raman spectroscopy that its imino tautomer occurs at a concentration of about half percent, more than two orders of magnitude greater than that of the canonical base Cytosine. Significantly, this proportion of unfavored tautomer is consistent with its enhanced tendency to induce C®T transition mutations relative to the natural base Cytosine. Our goal now is to demonstrate directly the imino tautomer of 5-hydroxy-Cytosine in a base mispair with Adenine within the active site of DNA polymerase.
We are attempting to develop a rationale for the organization and evolution of the genetic code, based in part upon an analysis of the energetics of codon-anticodon interaction and of the nonrandom nature of modern mRNA codon sequences. Toward those ends, we are investigating co-occurrence of particular groups of codons in protein messages, codon and amino acid usage bias, and other fossil remains of the earliest messages, notwithstanding mutation over eons of time. Recently we were able to develop evidence for those amino acids that entered the genetic code very early and those that entered it relatively late.
Alvarez-Dominguez JR, Amosova O, Fresco JR. (2013) Self-catalytic DNA depurination underlies human β-globin gene mutations at codon 6 that cause anemias and thalassemias. J Biol Chem. 288: 11581-9. PubMed
Amosova, O., Kumar, V., Deutsch, A. and Fresco, J.R. (2011) Self-catalyzed site-specific depurination of G-residues mediated by cruciform extrusion in closed circular DNA plasmids. J. Biol. Chem. 286: 36322–36330. Pubmed
Amosova, O., Smith, A. and Fresco, J.R. (2011) The consensus sequence for self-catalyzed site-specific G-residue depurination in DNA. J. Biol. Chem. 286: 36316-36321. Pubmed
Varganov Y, Amosova O, Fresco JR. (2006) Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells. Gene Ther 14: 173-179. PubMed
Amosova O, Coulter R, Fresco JR. (2006) Self-catalyzed site-specific depurination of guanine residues within gene sequences. Proc Natl Acad Sci 103: 4392-4397. PubMed
Liang H, Landweber LF, Fresco JR. (2005) Are stop codons recognized by base triplets in the large ribosomal RNA subunit? RNA 11: 1478-1484. PubMed
Broitman SL, Amosova O, Fresco JR. (2003) Repairing the Sickle Cell mutation. III. Effect of irradiation wavelength on the specificity and type of photoproduct formed by a 3'-terminal psoralen on a third strand directed to the mutant base pair. Nucleic Acids Res 31: 4682-4688. PubMed
Amosova O, Broitman SL, Fresco JR. (2003) Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence. Nucleic Acids Res 31: 4673-4681. PubMed
Johnson MD, 3rd, Fresco JR. (1999) Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei. Chromosoma 108: 181-189. PubMed
Broitman S, Amosova O, Dolinnaya NG, Fresco JR. (1999) Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair. J Biol Chem 274: 21763-21768. PubMed
Suen W, Spiro TG, Sowers LC, Fresco JR. (1999) Identification by UV resonance Raman spectroscopy of an imino tautomer of 5-hydroxy-2'-deoxycytidine, a powerful base analog transition mutagen with a much higher unfavored tautomer frequency than that of the natural residue 2'-deoxycytidine. Proc Natl Acad Sci USA 96: 4500-4505. PubMed