We have several broad goals and all focus on whether and to what extent stacking forces between nearest neighbor base pairs contribute to selective physical and biological properties of DNA. The most immediate, specific goal is the quantification of free energies of stacking interactions for the ten unique neighbor pairs. Sensitive measurements of stacking energies will be made from melting temperatures of sharp subtransitions for specially designed, multicopy, tandemly repeating synthetic inserts in polymeric plasmid DNAs, that exhibit readily detectable shifts in Tm in response to small variations in stacking energy. Microcalorimetric measurements will also be performed on these constructs in an attempt to measure the dependence of domain enthalpies on neighbor frequencies. The further goals of this project will then be to investigate selected sequence domains in both natural and synthetic sequences that exhibit anomalous thermal stabilities; to examine the relationship of stacking energies to local helical parameters determined by single crystal diffraction analysis; to explore for a dependence on stacking energies of patterns of repair of mismatched base pairs, as well as of mutation patterns and evolutionary patterns of selected sequence elements. Similar thermal stability measurements of stacking energies will be made on specific neighbors, similarly assembled in synthetic inserts in the form of repeats or tracts, to determine whether such tracts exhibit anomalous stabilities that can be attributed to next neighbor and beyond interactions in the duplex. In a related study, sensitive thermal stability studies will be conducted on reassociated recombinant plasmid-duplexes containing multicopy tandem repeats of synthetic inserts with specific mispairs. The objective of these studies is to measure the energetics of selected mispairs as well as the effects of the local sequence environment. These thermodynamic studies will be accompanied by sequencing studies to determine the nature of mispair correction in these inserts after transformation of E. coli cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022827-14
Application #
3271371
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1976-04-01
Project End
1994-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
14
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Maine
Department
Type
Schools of Earth Sciences/Natur
DUNS #
City
Orono
State
ME
Country
United States
Zip Code
04473
Qureshi, S A; Blake, R D (1995) Sequence characteristics of a cervid DNA repeat family. J Mol Evol 40:400-4
Marx, K A; Hess, S T; Blake, R D (1994) Alignment of (dA).(dT) homopolymer tracts in gene flanking sequences suggests nucleosomal periodicity in D. discoideum DNA. J Biomol Struct Dyn 12:235-46
Hess, S T; Blake, J D; Blake, R D (1994) Wide variations in neighbor-dependent substitution rates. J Mol Biol 236:1022-33
Marx, K A; Hess, S T; Blake, R D (1993) Characteristics of the large (dA).(dT) homopolymer tracts in D. discoideum gene flanking and intron sequences. J Biomol Struct Dyn 11:57-66
Blake, R D; Hess, S T; Nicholson-Tuell, J (1992) The influence of nearest neighbors on the rate and pattern of spontaneous point mutations. J Mol Evol 34:189-200
Delcourt, S G; Blake, R D (1991) Stacking energies in DNA. J Biol Chem 266:15160-9
Blake, R D; Delcourt, S G (1990) Electrostatic forces at helix-coil boundaries in DNA. Biopolymers 29:393-405
Blake, R D; Delcourt, S G (1988) Elasticity of DNA in nonhelical loops. Biochem Pharmacol 37:1843-4
Blake, R D; Delcourt, S G (1987) Loop energy in DNA. Biopolymers 26:2009-26
Blake, R D (1987) Cooperative lengths of DNA during melting. Biopolymers 26:1063-74

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