Professor; AB, Oberlin College; MA and PhD, Harvard University; NSF and NIH Fellow; Alfred P. Sloan Fellow; Josiah Macy, Jr. Foundation Faculty Scholar; Herbert Newby McCoy Research Award.

Research Description

Chemical nucleases are artificial catalysts which cleave the phosphodiester backbone of DNA and RNA by oxidative attack on the ribose moiety. Our laboratory discovered the first reagent of this type, the 2:1 1,10-phenanthroline-cuprous which acts with hydrogen peroxide as a coreactant. This simple coordindation complexes cleaves double-stranded DNA and single stranded regions of RNA. It is a very effective footprinting reagent and can be used in conjunction with gel retardation analysis to define the binding site of a protein or peptide on a nucleic acid that can be isolated by this widely used electrophoretic method.

One of the most interesting features of this simple coordination complex is its ability to react with the melted DNA formed when RNA polymerase generates a kinetically competent open complex with a promoter. This observation has led to the recognition that that tetrahedral 1,10-phenanthroline-cuprous complex binds to "transcription bubbles". We are exploiting this observation in the design of a new family of inhibitors of transcription which will reveal molecular details on the mechanism of transcription and generate pharmacologically active molecules.

A second phase of our work involves linking the nucleolytic activity to proteins, nucleic acids and DNA binding drugs. By attaching 1,10-phenanthroline-copper to these DNA binding ligands, highly selective site-specific nucleases can be produced. In one series of experiments, the E. Coli trp repressor has been converted into an efficient scission reagent capable of cleaving its high affinity binding sites. Mutagenesis is being used to alter the DNA binding specificity of the protein in order to produce a new series of specific reagents based on the trp repressor. In a second series of experiments, RNA's have been derivatized so that they are capable of cutting target DNAs in a sequence-specific fashion subsequent to the formation of R-loops. These reagents are being used to map chromosomal breaks points and the exon-intron structure of eucaryotic transcription units.

Keywords:

Bioorganic Chemistry: reaction mechanisms in molecular biology; chemical nuclease; gene-specific inhibitors; design of site-specific DNA and RNA scission reagents; chromosomal mapping.

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