A wide range of enzymes, including an increasing number of DNA repair enzymes, are known or suspected to use base flipping as a way to both recognize and access DNA in order to carry out further chemistry on the DNA bases. The DNA repair enzymes provide a robust line of defense against the loss of cellular functions due to DNA damage resulting in altered protein coding, as well as the accumulation of genetic mutations that may ultimately lead to cell death, tumors or cancer growth. In addition, as the work decoding the humangenome progresses, it is likely that other base flipping enzymes will be discovered that play key roles in DNA modification, repair, or gene expression and regulation. Thus, understanding the mechanism of base flipping is an important part of understanding and targeting therapies directed at diseases caused by the functioning or malfunctioning of these enzymes. Nucleoside analogs with a lower intrinsic rate of base flipping will be synthesized to use as a tool to probe the enzymatic mechanism of base flipping by Uracil DNA Glycosylase (UDG),a prototypical DNA repair enzyme. The synthesis of 5'-methyl nucleoside analogs of defined stereochemistry should hinder the bond rotations about the sugar phosphate backbone of DNA and slow down the rate of base flipping. It will be established that these analogs do indeed slow down the rate of base flipping by measuring the differences in the base pair lifetimes by NMR spectroscopy of double stranded DNA oligonucleotides containing the modified and unmodified nucleosides. Measurement of the longitudinal relaxation times (T1) of the imino protons as a function of the concentration of an added proton exchange catalyst (ammonia) will be used to determine the base pair lifetime. The modified nucleosides should increase the base pair lifetime which is equivalent to reducing the rate of base pair flipping. Finally, the nucleoside analogs will be put to the test with UDG to see how the altered rate of base flipping affects the enzyme kinetics. Enzymes, like UDG, that use a base flipping mechanism should show different profiles, depending on how and when base flipping occurs. Ultimately, this methodology should be useful for studying base flipping in a variety of enzymes.
