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Dissertation
Studies on DNA photolyase from Escherichia coli and Saccharomyces cerevisiae
Doctor of Philosophy (Ph.D.), Allegheny University of the Health Sciences
Jul 1996
DOI:
https://doi.org/10.17918/00009850
Abstract
DNA Photolyase catalyzes the direct photorepair of the cyclobutane dimer between adjacent pyrimidines, the main product of DNA exposed to ultraviolet light. The native enzymes from both Escherichia coli and Saccharomyces cerevisiae contain a folate chromophore ( (6R) -5, 10-CH+-H4Pte(Glu)n, n = 3-6) and a fully reduced flavin (FADH2). In E. coli photolyase, the folate acts as an antenna to transfer light energy to the FADH2 at a high efficiency (EET=0.92). Apophotolyase reconstituted after an overnight incubation with (6-R,S) -5, 10-CH+-H4folate, a monoglutamate analog of the native cofactor) contains equimolar amounts of the [6R]- and [6S]-isomers, suggesting similar binding affinities. A rapid biphasic increase in fluorescence ([approx equals]100-fold) is observed upon binding of 5,10-CH+-H4folate to apophotolyase ar 5 [degrees] C; the [6S]-isomer binds about 25-fold faster than the [6R]-isomer. Although identical absorption and fluorescence emission maxima are observed for enzyme reconsitituted with [6R]-, [6S]-, or [6-R,S]-5,10-CH+-H4folate, folate fluorescence quantum yield values vary depending on the stereochemical configuration at the 6 position ([Phi]=0.82, 0.18, or 0.46, respectively, at 5 [degrees]C), a feature not seen with free folate. The fluorescence of enzyme-bound folate is quenched upon flavin binding; the quenching efficiency by flavin radical (EQ=0.96) or FADH2 (EQ=0.89) is the same for both folate isomers. In contrast, energy transfer from folate to FADH2 is sensitive to the stereochemical configuration at the 6 position. The efficiency of energy transfer observed for enzyme containing FADH2 and [6R]-, or [6S]-, or [6-R,S]-5, 10-CH+-H4folate (0.66, 0.26, or 0.44, respectively) is directly proportional to the fluorescence quantum yield observed for folate in the absence of FADH2, as expected for Forster-type energy transfer. Although less efficient, the unnatural [6S]-isomer is catalytically functional, a feature not previously observed with other folate-dependent enzymes. Fluorescence quantum yield studies at 77 K with free ([Phi]=0.67) and enzyme-bound ([Phi]=1.0) folate suggest that differences in solvent exposure may contribute to the fluorescence efficiency differences observed with the enzyme-bound folate isomers at 5 [degrees] C. Expression of yeast photolyase from the existing tac expression system, using the pCB1241 plasmid, produced only modest yields of isolable enzyme (0.13 mg enzyme per gram of cells) due to proteolysis and the presence of insoluble aggregates. An improved expression system for yeast photolyase has been constructed utilizing the highly specific T7 promoter and an expression strain lacking the major E. coli proteases. Using the new plasmid, pRL100, larger quantities of recombinant enzyme have been isolated (2.1 mg/g of cells), free from proteolytic degradation, in a yield 15-fold greater than the earlier expression system. The availability of substantial amounts of pure yeast photolyase has permitted preliminary crystallographic studies.
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Details
- Title
- Studies on DNA photolyase from Escherichia coli and Saccharomyces cerevisiae
- Creators
- Richard Simon Adam Lipman
- Awarding Institution
- Allegheny University of the Health Sciences
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Allegheny University of the Health Sciences; Philadelphia, Pennsylvania
- Number of pages
- x, 144 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry [Historical]; Allegheny University of the Health Sciences (1996-1998); School of Medicine (1996-1998)
- Other Identifier
- 991021888827604721