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 (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

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.
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PMID:Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis. 353 40

The absolute action spectrum of Escherichia coli DNA photolyase was determined in vitro. In vivo the photoreactivation cross-section (epsilon phi) is 2.4 X 10(4) M-1 cm-1 suggesting that the quantum yield (phi) is about 1.0 if one assumes that the enzyme has the same spectral properties (e.g. epsilon 384 = 1.8 X 10(4) M-1 cm-1) in vivo as those of the enzyme purified to homogeneity. The relative action spectrum of the pure enzyme (blue enzyme that contains FAD neutral semiquinone radical) agrees with the relative action spectrum for photoreactivation of E. coli, having lambda max = 384 nm. However, the absolute action spectrum of the blue enzyme yields a photoreactivation cross-section (epsilon phi = 1.2 X 10(3) at 384 nm) that is 20-fold lower than the in vivo values indicative of an apparent lower quantum yield (phi approximately equal to 0.07) in vitro. Reducing the enzyme with dithionite results in reduction of the flavin semiquinone and a concomitant 12-15-fold increase in the quantum yield. These results suggest that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300-500 nm range, the photolyase chromophore (which consists of reduced FAD plus the second chromophore) donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. The reduced chromophore is regenerated at the end of the photochemical step thus enabling the enzyme to act catalytically.+
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PMID:Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum. 353 41

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.
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PMID:UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells. 354 May 89

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.
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PMID:Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts. 354 34

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 nucleotide sequence of a 2301 base pair region of Saccharomyces cerevisiae DNA containing the PHR1 gene is reported. Within this region a single open reading frame of 1695 base pairs was found; using the insertional inactivation technique it was shown that part or all of this open reading frame specifies the PHR1-encoded photolyase. The amino acid sequence of the 565 amino acid long polypeptide predicted from the PHR1 nucleotide sequence was compared to the amino acid sequence of E. coli photolyase. Overall the sequence homology was 36.5%; however, two short regions near the amino terminus as well as the carboxy-terminal 150 amino acids display significantly greater sequence homology. The presence of these strongly conserved regions suggests that the yeast and E. coli photolyase possess common structural and functional domains involved in substrate and/or chromophore binding.
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PMID:Sequence of the Saccharomyces cerevisiae PHR1 gene and homology of the PHR1 photolyase to E. coli photolyase. 390 69

DNA photolyase from Escherichia coli is a blue protein exhibiting absorption maxima at 580, 475, and 384 nm. One of the two chromophores present in this enzyme has been identified as the blue neutral flavin adenine dinucleotide (FAD) radical on the basis, in part, of visible absorption and electron spin resonance (ESR) data. The enzyme-bound radical (epsilon 580 = 3.6 X 10(3) M-1 cm-1) is stable toward O2 or K3Fe(CN)6, is reversibly reduced by dithionite, and is converted to oxidized FAD upon aerobic denaturation. Disproportionation of the radical is observed upon anaerobic denaturation, consistent with an N-5 unsubstituted radical. The absorbance of the enzyme at lambda greater than 500 nm is due only to the FAD radical whereas the band at 384 nm reflects contributions from both the radical and a second chromophore. The latter is labile when protein free a neutral pH (lambda max = 360 nm, k = 5.5 X 10(-2) min -1 +/- O2), a reaction that is readily monitored by the loss of an intense absorption band at 360 nm following enzyme denaturation under conditions where radical oxidation is immediate. This decomposition is pH dependent and the chromophore is stable at acid pH. Native photolyase is fluorescent (emission lambda max = 470 nm, excitation lambda max = 398 nm). An unlikely fluorescent flavin radical can be excluded by the position of the emission maximum. The enzyme fluorescence is attributed to the second chromophore.
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PMID:Identification of a neutral flavin radical and characterization of a second chromophore in Escherichia coli DNA photolyase. 608 79

One of the principal photochemical reactions of DNA on exposure to UV is the formation of intrastrand cyclobutane-type pyrimidine dimers. The efficiency of this reaction depends on both the wavelength of the UV2 and the specific nucleotide sequence in the DNA. The formation of the pyrimidine dimer and its repair in living cells have been studied extensively. We have examined the possibility that pyrimidines at the ends of DNA strands may be adequately juxtaposed for dimer formation by the presence of a complementary strand, even when no phosphodiester linkage joins their sugars. In these conditions the formation of a dimer will 'ligate' two DNA strands end-to-end. We report here that thymidine oligonucleotides annealed to polydeoxyadenylate can be ligated end-to-end by UV irradiation, via thymine dimerization of the terminal nucleotides in adjacent oligonucleotides. The linkages are susceptible to direct photoreversal by 254 nm UV, as expected for cyclobutane-type thymine dimers, but they are not cleaved by the bacteriophage T4 endonuclease V, a dimer-specific DNA repair enzyme. We demonstrate that the ligating dimers are also resistant to photolyase from Escherichia coli. Although the phosphodiester backbone is not required for dimer formation, it is required for recognition of dimers by these DNA repair enzymes. We discuss the possibility that high molecular weight polynucleotides in primordial seas might have been generated from oligonucleotides by pyrimidine dimerization under the intense solar UV flux unattenuated by an ozone layer.
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PMID:Ligation of oligonucleotides by pyrimidine dimers--a missing 'link' in the origin of life? 628 88

We have constructed a series of multicopy plasmids that complement mutations in the phr gene of Escherichia coli. By subcloning into a tac plasmid vector we obtained a phr plasmid that upon induction overproduces two proteins of Mr's 49,000 and 20,000. Tn1000 insertions into the phr gene caused the disappearance of the 49,000 dalton protein, thus demonstrating this protein to be the phr gene product, DNA photolyase. The photolyase encoded by the phr gene makes up about 15% of total cellular proteins after induction of cells carrying a tac-phr plasmid. This protein binds specifically to UV (254 nm) irradiated DNA and upon exposure to near UV (300-500 nm) illumination repairs the UV damage and dissociates from DNA.
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PMID:Identification and amplification of the E. coli phr gene product. 631 52


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