Characterizing the Structure and Function of E-DNA

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Ho, PuiShingMCB-0090615This research is designed to characterize the conformational determinants and the potential significance of a new form of the DNA double-helix induced by cytosine methylation (d m5C) or bromination (d Br5C). This novel conformation was discovered in the single-crystal structure of d(GGCGm5CC)2 and d(GGCGBr5CC)2 . The obvious questions that arise with any new conformation of DNA discovered by X-ray crystallography include: 1) what are the sequence requirements for its formation, 2) does this form exist in solution, and 3) is the structure relevant? The long-term goal is to use this structural information to locate E-DNA ingenomic sequences and, consequently, place the conformation in a broader biological context.The first objective of this project is to define the types of sequences beyond the parent sequence d(GGCGm5CC)2 that can adopt this novel conformation. The second objective is to determine whether E-DNA exists in solution by defining a spectroscopic signature that links the conformation in the crystal with its formation in aqueous solution. The third objective is to test the hypothesis that E-DNA is a discrete intermediate in the pathway that leads to A-DNA. The studies will start with the complete set of crystal structures from B-DNA to A-DNA for the sequence motif d(GGCGCC)2 , and use these structures as a basis for molecular simulations to map the thermodynamics for the B-DNA to A-DNA transition. The results will be confirmed by stopped-flow transient kinetic studies. The final objective is to test the hypothesis that the conformation and solvent structure of E-DNA facilitates the deamination reaction that ultimately leads to the rapid mutation of dm5C to dT nucleotides. The incorporation of 18O from isotopically enriched water into the crystals of A-DNA, B-DNA, and E-DNAwill be monitored.