It has been proposed that ion-induced quadruplex formation could be involved in telomere-telomere interactions, chromosomal pairing, recombination, and repression of transcription. Changes in telomeric DNA length correlate with changes in chromosome maintenance, cellular senescence and genetic diseases. Results suggest that recognition of G- rich DNAs by telomerases, transcription factors or polymerases and recombinases may be reversibly suppressed, or enhanced in the case of recombinases, by quadruplex formation.
The specific aims are : 1) To study quadruplex dissociation kinetics from the point of view of both the 'host' DNA and the 'guest' ions. CD strand-dissociation kinetics data will be analyzed and the reaction order will be determined. DNA strand concentration, ion concentration and ion type, pH and temperature will be varied then dissociation constants and transition state free energies will be determined. The effect of cytosine methylation on pH- dependent quadruplex stabilization will also be assessed. 2) Investigate the use of terbium (Tb3+) fluorescence analysis to monitor equilibrium ion binding and dissociation reaction kinetics from the point of view of the ion. These measurements will provide a sensitive method to characterize ion / DNA stoichiometries and kinetics of ion-induced changes in telomeric DNAs at much lower concentrations than previously accomplished paving the way for use as a probe for quadruplexes in situ. 3) Produce and develop the use of antibodies as tools to assess whether G-DNA quadruplexes are involved in telomeric functions and interchromosomal interactions. We will generate monoclonal antibodies that recognize well-characterized DNA quadruplex complexes and characterize the specificity of these antibodies for the quadruplex conformation as a function of solution conditions (specific ions, temperature and pH) and relative to other non-quadruplex structures (B- ,Z-,A-, triplex and single stranded forms). We will then test these antibodies for specific recognition of previously characterized quadruplex structures formed by telomeric DNAs from Tetrahymena, yeast and humans, and whether they form in yeast artificial chromosomes in vitro or in vivo. 4) Since the antibodies should bind tighter to the quadruplex, antibodies or FABs may catalyze quadruplex formation. We will determine whether this occurs and, if so, whether this mechanism promotes transcriptional repression or DNA strand associations. 5) To use these antibodies in immunofluorescence microscopy experiments to probe for the biological occurance and localization of quadruplex structures in situ and in vivo. The long range goals are to use these probes to study synaptic chromosomal complexes and mutant chromosomes produced by translocation events. Deleterious chromosomal translocations have been correlated with dysfunctional aspects of B-cell differentiation and tumorigenesis. The reagents developed in the proposed studies may help to clarify these issues by linking specific DNA sequences to a structural mechanism for faulty chromosomal translocation events.
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