STRUCTURE AND INTERACTIONS OF DNA

本研究的长远目标是评估

The distant objectives of this study are the evaluations of thermodynamic characteristics associated with selected nonhelical and aberrant structures of DNA, some that may provide specificity for the attraction of enzymes for replication, transcription, etc. Quantitative appreciation of the forces that induce different modes of dynamic behavior in DNA should help explain subtle effects on gene expression of small changes in sequence in noncoding regions, as well as the evolution of DNA sequences. The near-term goal is to measure the energetic costs of interrupting the helix and maintaining nonhelical loop structures, including the shear forces needed to separate opposing chains over short lengths in internal stretches of helix. Initially it is intended that the loop forming specimen be short lengths of oligo(A degrees T) N, where 50 greater than N greater than 10 bp, sandwiched between stable boundaries of (G degrees C) 40-80 degrees. The specimen will be synthesized as a recombinant in the Pst I site of pBR322 and excised for the physical studies. Thermodynamic characteristics are to be extracted from observations of the helix-nonhelix equilibrium monitored by high resolution optical melting at several wavelengths simultaneously by the digital-difference approximation method. Later studies will take up the question of loop formation in domains of different base compositions and sequence features. In a correlated study the thermodynamics associated with each of the ten unique stacking interactions will be evaluated from the stabilities of specific, well characterized domains producing the multitransitional melting curve of pBR322. Nearest neighbor frequencies, to which the ten stability constants are related, will be assigned to specific subtransitions that reveal themselves by changes in melting temperature when the domains responsible for the subtransitions are cut by restriction enzymes. In a separate study, the precise amounts and level of sequence variation will be determined for the 1399bp satellite I and 46bp satellite II repetitive sequences from the bovine genome. Sequence variations in these satellites will be determined from the increase in the range of temperature of the subtransition for the population of repetitive sequences over that of single cloned specimens.