Dissertation

The action mechanism of Escherichia coli DNA photolyase

Payne, Gillian

1990

Overview

Escherichia coli DNA photolyase mediates the photocatalyzed repair of cyclobutane pyrimidine dimers occurring in UV-irradiated DNA. The binding of photolyase to dimer substrate has been investigated using flash photolysis and the transformation assay. The binding association constant, k$\\sb1$, was found to be 1.4-4.2 $\\times$ 10$\\sp6$ M$\\sp{-1}$cm$\\sp{-1}$. The dissociation of enzyme from substrate was biphasic with k$\\sb{\\rm 2a}$ = 2-3 $\\times$ 10$\\sp{-2}$ s$\\sp{-1}$ and k$\\sb{\\rm 2b}$ = 0.6-1.3 $\\times$ 10$\\sp{-3}$ s$\\sp{-1}$. E. coli photolyase, when purified, contains a neutral blue radical FAD chromophore (FADH$\\sp\\circ$). However, EPR measurements on whole E. coli cells of the photolyase overproducing strain indicated that the FAD is fully reduced (FADH$\\sb2$) in vivo and that enzyme containing FADH$\\sp\\circ$ is the result of a purification artifact. Photoreduced enzyme (E-FADH$\\sb2$) repaired dimers with a greater efficiency than FADH$\\sp\\circ$. Activity measurements using long wavelength photoreactivating light indicated that E-FADH$\\sp\\circ$ is not active. In addition to FADH$\\sb2$, photolyase contains 5,10-methenyltetrahydro-folylpolyglutamate (MTHF). Action spectrum measurements conducted with E-FADH$\\sb2$ and with E-FADH$\\sb2$-MTHF indicated that E-FADH$\\sb2$ is catalytically competent but that E-FADH$\\sb2$-MTHF is more active. The quantum yield of electron transfer, $\\Phi\\sb{\\rm ET}$ of E-FADH$\\sb2$ = 0.69 while that of E-FADH$\\sb2$-MTHF is 0.59. These $\\Phi\\sb{\\rm ET}$ are essentially wavelength-independent. We propose that MTHF \"transfers\" energy to FADH$\\sb2$ with a quantum yield of energy transfer ($\\Phi\\sb{\\rm ET}$) of 0.80 and that FADH$\\sb2$ transfers an electron to or from the dimer with $\\Phi\\sb{\\rm ET}$ = 0.69 to result in an overall $\\Phi\\sb{\\rm ET}$ of 0.59 for E-FADH$\\sb2$-MTHF. Furthermore, $\\Phi\\sb{\\rm ET}$(E-FADH$\\sb2$-MTHF) was found to be the same for thymine dimers in five different duplex sequence contexts and in single-stranded DNA. In studies where both chromophores are reversibly removed from the enzyme, the apoenzyme was found to have no affinity to thymine dimer-containing DNA. However, when reconstituted with FAD or 5-deazaFAD, the enzyme recovered substrate binding activity indicating that the FAD helps to form the substrate binding pocket. The failure of FMN, riboflavin, 1-deazaFAD, and F$\\sb{420}$ to bind to apophotolyase indicates that the N1 and N10 substituent of the flavin help to determine the chromophore binding specificity.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry
• ISBN: 9798207775067