Mary E. Hatcher

31P NMR Investigation of Backbone Dynamics in DNA Binding Sites

The backbone conformation of DNA plays an important role in the indirect readout mechanisms for protein--DNA recognition events. Thus, investigating the backbone dynamics of each step in DNA binding sequences provides useful information necessary for the characterization of these interactions. Here, we use 31P dynamic NMR to characterize the backbone conformation and dynamics in the Dickerson dodecamer, a sequence containing the EcoRI binding site, and confirm solid-state 2H NMR results showing that the C3pG4 and C9pG10 steps experience unique dynamics and that these dynamics are quenched upon cytosine methylation. In addition, we show that cytosine methylation affects the conformation and dynamics of neighboring nucleotide steps, but this effect is localized to only near neighbors and base-pairing partners. Last, we have been able to characterize the percent BII in each backbone step and illustrate that the C3pG4 and C9pG10 favor the noncanonical BII conformation, even at low temperatures. Our results demonstrate that 31P dynamic NMR provides a robust and efficient method for characterizing the backbone dynamics in DNA. This allows simple, rapid determination of sequence-dependent dynamical information, providing a useful method for studying trends in protein-DNA recognition events.

Dioxygen diffusion in the stratum corneum: an EPR spin label study

The stratum corneum, the outer 10 microns of the skin, serves as a permeability barrier regulating the transport of molecules between the body and the environment. The purpose of this study is to understand this permeability barrier function as it pertains to the diffusion of molecular oxygen. The stratum corneum was investigated with EPR spectroscopy following inoculation with a stearic acid spin probe. The presence of paramagnetic molecular oxygen results in the broadening of the EPR spectral lines of the spin probe. The rate of oxygen diffusion across the stratum corneum, and then the oxygen diffusion coefficient, D(O2), was determined by studying this line-broadening as a function of time. D(O2) in human stratum corneum was found to be 3 x 10(-7) cm2/s at 37 degrees C with an activation energy of approx. 44 kJ/mol. The application of the permeation-enhancing chemicals, DeMSO and DMSO, to the stratum corneum increased D(O2) two- to three-fold.

An FTIR Investigation of Flanking Sequence Effects on the Structure and Flexibility of DNA Binding Sites

Fourier transform infrared (FTIR) spectroscopy and a library of FTIR marker bands have been used to examine the structure and relative flexibilities conferred by different flanking sequences on the EcoRI binding site. This approach allowed us to examine unique peaks and subtle changes in the spectra of d(AAAGAATTCTTT)(2), d(TTCGAATTCGAA)(2), and d(CGCGAATTCGCG)(2) and thereby identify local changes in base pairing, base stacking, backbone conformation, glycosidic bond rotation, and sugar puckering in the studied sequences. The changes in flanking sequences induce differences in the sugar puckers, glycosidic bond rotation, and backbone conformations. Varying levels of local flexibility are observed within the sequences in agreement with previous biological activity assays. The results also provide supporting evidence for the presence of a splay in the G(4)-C(9) base pair of the EcoRI binding site and a potential pocket of flexibility at the G(4) cleavage site that have been proposed in the literature. In sum, we have demonstrated that FTIR is a powerful methodology for studying the effect of flanking sequences on DNA structure and flexibility, for it can provide information about the local structure of the nucleic acid and the overall relative flexibilities conferred by different flanking sequences.