Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.99.3 (PRE)
1,923 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli DNA photolyase is a flavoprotein that when purified is blue in color and contains a stable neutral radical FAD (E-FADH). In the presence of a suitable electron donor (i.e., thiols, tyrosine, or NADH) the radical FAD adsorbs visible light and undergoes photoreduction to the fully reduced FAD (E-FADH2). The in vitro quantum yield of dimer repair for E-FADH is 0.07 while that of E-FADH2 approaches the in vivo value of 1. Electron paramagnetic resonance studies on whole cells indicate that the in vivo form of photolyase is E-FADH2 with enzyme containing radical FAD generated predominantly during the ammonium sulfate precipitation step of the purification. Activity measurements of E-FADH using long-wavelength photoreactivating light indicate that enzyme containing FAD in the radical form is not active in dimer repair. Dimer repair observed with E-FADH at shorter wavelengths is probably photoreduction of E-FADH followed by dimer repair by E-FADH2.
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PMID:The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro. 282 44

A 2 kb DNA fragment, containing the photoreactivation gene phr1 from Escherichia coli, was inserted at the BamH1 site in the tet gene of the yeast--E. coli shuttle vector pJDB207. Photoreactivation--deficient Saccharomyces cerevisiae cells transformed with this plasmid showed photoreactivation of killing after UV irradiation of the cells, while extracts of transformed cells exhibited photoreactivating activity in vitro. Far more photoreactivating enzyme molecules were found when the gene was inserted in the plasmid in the opposite orientation to the tet gene as compared with a plasmid carrying the inserted gene in the same orientation. Photoreactivating enzyme encoded by the E. coli phr1 gene and produced in transformed yeast cells has characteristics of the E. coli photoreactivating enzyme (flavoprotein) as judged from the influence of ionic strength on photoreactivating activity.
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PMID:Expression of an Escherichia coli phr gene in the yeast Saccharomyces cerevisiae. 299 90

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.
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PMID:Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex. 353 39

The E. coli DNA photolyase is a flavoprotein that catalyzes the photoreversal of pyrimidine dimers. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and repairs the dimer upon absorbing a photon in the 300-600 nm range. The rate and equilibrium constants for the light-independent reaction were determined before, using randomly modified substrates that contained T mean value of T, T mean value of C and C mean value of C dimers in random sequence surrounding. In this paper we have determined these constants for a defined substrate (a 43 bp oligomer containing a T mean value of T dimer) using the gel retardation assay. We find that: the equilibrium constant and the off rate obtained with this substrate by this technique are similar to those obtained with randomly modified DNA using filter binding and flash photolysis techniques. the off rate with the defined substrate is heterogeneous indicating heterogeneity in the enzyme population or in the enzyme-substrate complexes, and the enzyme has 7.5 X 10(4)-fold higher affinity for pyrimidine dimer compared to non-dimer DNA nucleotides.
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PMID:Binding of E. coli DNA photolyase to a defined substrate containing a single T mean value of T dimer. 354 32

Escherichia coli DNA photolyase is a flavoprotein which catalyzes the photomonomerization of pyrimidine dimers produced in DNA by UV irradiation. In vivo, the enzyme acts by a two-step mechanism: it binds to dimer-containing DNA in a light-independent reaction and upon exposure to 300-500-nm light breaks the cyclobutane ring and dissociates from the substrate. Using photolyase purified to homogeneity, we have investigated in vitro the first step of the reaction, DNA binding; enzyme-DNA complex formation was quantitated by the nitrocellulose filter binding assay. We find that the enzyme binds specifically to UV-irradiated DNA regardless of whether the DNA is in the superhelical, open circular, or linear form or whether the DNA is single or double stranded. The binding reaction is optimum at a NaCl concentration of 125 mM and at pH 7.5. Although photolyase is retained by the nitrocellulose filters with near 100% efficiency, the binding efficiency of a single enzyme-substrate complex is about 0.34. The complexes can be dissociated by exposing them to photoreactivating light either in solution or on the filter.
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PMID:Binding of Escherichia coli DNA photolyase to UV-irradiated DNA. 389 38

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.
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PMID:Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1. 763 20

DNA photolyase is a flavoprotein that repairs cyclobutylpyrimidine dimers by ultrafast photoinduced electron transfer. One unusual feature of this enzyme is the configuration of the FAD cofactor, where the isoalloxazine and adenine rings are nearly in vdW contact. We have measured the steady-state and transient absorption spectra and excited-state decay kinetics of oxidized (FAD-containing, folate-depleted) Escherichia coli DNA photolyase with and without dinucleotide and polynucleotide single-stranded thymidine dimer substrates. The steady-state absorption spectrum for the enzyme-polynucleotide substrate complex showed a blue shift, as seen previously by Jorns et al. (1). No shift was observed for the dinucleotide substrate, suggesting that there are significant differences in the binding geometry of dinucleotide versus polynucleotide dimer lesions. Evidence was obtained from transient absorption experiments for a long-lived charge-transfer complex involving the isoalloxazine of the FAD cofactor. No evidence of excited-state quenching was measurable upon binding either substrate. To explain these data, we hypothesize the existence of a large substrate electric field in the cavity containing the FAD cofactor. A calculation of the magnitude and direction of this dipolar electric field is consistent with electrochromic band shifts for both S(0) --> S(1) and S(0) --> S(2) transitions. These observations suggest that the substrate dipolar electric field may be a critical component in its electron-transfer-mediated repair by photolyase and that the unique relative orientation of the isoalloxazine and adenine rings may have resulted from the consequences of the dipolar substrate field.
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PMID:Evidence of powerful substrate electric fields in DNA photolyase: implications for thymidine dimer repair. 1173 3

DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH(-)) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 +/- 20 ps for the pentameric substrate. Repair of the carbon-carbon double bonds (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH(*) cofactor is not reduced to its catalytically active FADH(-) state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.
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PMID:Cis-syn thymidine dimer repair by DNA photolyase in real time. 1285 3

The 266 kbp genome sequence of plaque-purified, tissue culture-adapted, attenuated European Fowlpox virus FP9 has been determined and compared with the 288 kbp sequence of a pathogenic US strain (FPVUS). FP9 carries 244 of the 260 reported FPVUS ORFs (both viruses also have an unreported orthologue of conserved poxvirus gene A14.5L). Relative to FPVUS, FP9 differed by 118 mutations (26 deletions, 15 insertions and 77 base substitutions), affecting FP9 equivalents of 71 FPVUS ORFs. To help to identify mutations involved in adaptation and attenuation, the virulent parent of FP9, HP1, was sequenced at positions where FP9 differed from FPVUS. At 68 positions, FP9 and HP1 sequences were identical, reflecting differences between American and European lineages. Mutations at the remaining 50 positions in FP9 relative to FPVUS and HP1, involving 46 ORFs, therefore accounted for adaptation and attenuation. ORFs deleted during passage included those encoding members of multigene families: 12 ankyrin repeat proteins, three C-type lectin-like proteins, two C4L/C10L-like proteins, one G-protein coupled receptor protein, one V-type Ig domain protein, two N1R/p28 proteins and one EFc family protein. Tandem ORFs encoding Variola virus B22R orthologues were fused by a 5.8 kbp deletion. Single-copy genes disrupted or deleted during passage included those encoding a homologue of murine T10, a conserved DNA/pantothenate metabolism flavoprotein, photolyase, the A-type inclusion protein and an orthologue of vaccinia A47L. Gene assignments have been updated for DNase II/DLAD, binding proteins for IL-18 and interferon-gamma, phospholipid hydroperoxide glutathione peroxidase (PHGPX/GPX-4) and for a highly conserved homologue of ELOVL4.
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PMID:Comparison of the genome sequence of FP9, an attenuated, tissue culture-adapted European strain of Fowlpox virus, with those of virulent American and European viruses. 1476 88

A cryogenic optical waveguide spectrometer that uses a Teflon-AF 2400 liquid core waveguide is described. In comparison to standard low-temperature absorption techniques, the liquid core waveguide approach not only affords the use of microliter samples but also provides significant improvements in sensitivity. Here we show low-temperature absorption spectra of various flavoproteins, including DNA photolyase, measured using this new technique. The technique has high reproducibility and can afford the detection of 15 ng of flavoprotein. In addition, the technique requires several hundredfold less protein than standard low-temperature techniques for the same sensitivity. The performance of the spectrometer in the ultraviolet (UV) region is investigated experimentally and compared with standard UV absorption techniques. Results indicate that, below 300 nm, the observed absorbances deviate from the Beer-Lambert law.
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PMID:A cryogenic optical waveguide spectrometer for the measurement of low-temperature absorption spectra of dilute biological samples. 1564 84


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