Search

Research was conducted to determine the hydrogen abstraction event that starts the cleavage process from the sugar moiety. The formation of a strand break and hydrogen atoms is considered in the elucidating mechanisms of nucleic acid degradation. A representative set of DNA-cleaving pathways that is organized by an initial hydrogen abstraction event was described. The pathways were also determined by monitoring various unique marker products.

The effects of 2-methyl-2,4-pentanediol (MPD) precipitating agent on the crystal structure of the A-tract-containing (A5N5) deoxyribonucleic acid (DNA) was analyzed by utilizing hydroxyl radicals. MPD alters the hydroxyl radical cleavage pattern of the A5N5 DNA sequence into a mixed-sequence unbent DNA. However, the hydroxyl radical cleavage patterns of straight A-tract sequence or T4A4N2 sequence and mixed-sequence DNA was not affected by MPD.

Dihydrothiophene equilibrium binding is attained before the occurrence of strand scission and the free energy differences relative to the TCCT site are calculated. To compare the amount of stabilization in the binding of CLMgama(super 1)(sub 1) and CLMepsilon at a TCCT-AGGA sequence, using isothermal titration calorimetry and quantitative hydroxyl radical footprint titration. These reaction rates are studied to get an overall estimate of the energetics involved in the calicheamicin binding to DNA and in the role played by the side-chain aryl iodide.

The hydroxyl radical is responsible for DNA strand cleavage in the reaction of (FeIIEDTA)2- and H2O2 with DNA, rather than a high-valent iron-oxo complex. DNA cleavage patterns induced by gamma radiolysis and by the reaction of (FeIIEDTA)2- and H2O2 in the presence of ascorbate are identical with the DNA cleavage pattern produced by the hydroxyl radical.

Studies on the binding of calicheamicin (CLM) epsilon with DNA reveal CLM's hydroxyl radical footprinting. A comparison of CLM epsilons binding to DNA with DNA's binding by native CLM gamma reveals that CLM epsilon molecules are oriented to the DNA strands in an extended structure. These results validate the theory that global CLM structures will have the oligosaccharide side chain of the C5' hydrogen abstraction's 3' side.

A specific phosphodiester bond in DNA was cleaved using hydrogen peroxide in combination with the Flp recombinase obtained from Saccharomyces cerevisiae. In this system, cleavage resulted from peroxide's nucleophile action on a phosphorus oxygen bond rather than from oxidative damage of the DNA backbone. Under normal circumstances, the reaction would take place at a Flp binding site not damaged by the enzyme. Preoxide-mediated cleavage also occurred with an Flp mutant in which phenylalanine replaced the active site tyrosine.

A study was conducted on DNA structure in a nucleosome core at high salt concentration and temperature using hydroxyl radical cleavage of DNA as probe. Results reveal that a DNA fragment's rotational and translational positioning on the histone octamer is retained at NaCl concentrations of0.0-8.0 M and temperatures of 0-75 degrees centigrade. It was also found that at high salt concentrations, the helical periodicity observed in the DNA cleavage pattern in a nucleosome core at 0.0 M NaCl is retained.

The cleavage patterns of several TnAn sequences was obtained to characterize the structure of such sequences in DNA. A new structural model is proposed which explains its bending and local conformational properties. It is proposed that the core of TnAn sequences are B-DNA-like with adenines and thymines localized outside the core. However, the presence of sufficient number of A and T residues induces the formation of a structure that resembles that of adenine tracts surrounded by a mixed sequence DNA.

Calicheamicin-homeodomain conjugate is found to be an effective sequence-specific DNA cleavage and mapping tool.

Research is presented concerning the use of gamma-radiation for the production of the hydroxyl radical in order to reveal the structure of protein-DNA molecules at a resolution of a single nucleotide.