Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex

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.     

Active site of Escherichia coli DNA photolyase: mutations at Trp277 alter the selectivity of the enzyme without affecting the quantum yield of photorepair

Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction. The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA. No structural information on the enzyme is available at present. However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology. These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis. Trp277 (W277) in E. coli photolyase is conserved in all photolyases sequenced to date. We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis. Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis. Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties.     

Mechanism of damage recognition by Escherichia coli DNA photolyase

Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.     

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.     

Effect of base, pentose, and phosphodiester backbone structures on binding and repair of pyrimidine dimers by Escherichia coli DNA photolyase.

Photolyases reverse the effects of UV light on cells by converting cyclobutane dipyrimidine photoproducts (pyrimidine dimers, Pyr mean value of Pyr) into pyrimidine monomers in a light-dependent reaction. Previous work has suggested that, based on substrate preference, there are two classes of photolyase: DNA photolyase as exemplified by the Escherichia coli enzyme, and RNA photolyases found in plants such as Nicotiana tabacum and Phaseolus vulgaris. In experiments aimed at identifying substrate determinants, including the pentose ring, for binding and catalysis by E. coli DNA photolyase we tested several Pyr mean value of Pyr. We found that the enzyme has relative affinities for photodimers of T mean value of T greater than or equal to U mean value of T greater than U mean value of U much greater than C mean value of C and that the E-FADH2 form of the enzyme repairs these dimers at 366 nm with absolute quantum yields of 0.9 (T mean value of T), 0.8 (U mean value of T), 0.6 (U mean value of U), and 0.05 (C mean value of C). The enzyme also repairs an isolated thymine dimer and the synthetic substrate, 1,1'-trimethylene-bis (thymine) cyclobutane dimer. Unexpectedly, we found that this enzyme, previously thought to be specific for DNA, repairs uracil cyclobutane dimers in poly(rU). The affinity of photolyase for a uracil dimer in RNA is about 10(4)-fold lower than that for a U mean value of U in DNA; however, once bound, the enzyme repairs the photodimer with the same quantum yield whether the dimer is in ribonucleoside or deoxyribonucleoside form.     

The folate cofactor of Escherichia coli DNA photolyase acts catalytically

Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.     

Interaction of contractile proteins with DNA

The interaction of contractile proteins (myosin, actin, tropomyosin and troponin) with DNA was studied in vitro using a nitrocellulose filter binding technique. The data indicate a high affinity of myosin and troponin for DNA, a lesser interaction between DNA and tropomyosin and the absence of binding of actin to DNA. When binding to DNA was detected, the interaction was higher with single-stranded DNA than with RNA or double-stranded DNA, although in some conditions myosin binds equally as well to native as to denatured eukaryotic DNA. Myosin binds better to eukaryotic than to phage native DNA.     

Binding of E. coli DNA photolyase to a defined substrate containing a single T mean value of T dimer.

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.     

A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae.

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.     

A DNA-binding protein from Ustilago maydis prefers duplex DNA without chain interruptions.

Using a nitrocellulose filter binding assay, we have partially purified a protein from mitotic cells of Ustilago maydis that binds preferentially to covalently closed circular duplex DNA. DNA containing single- or double-strand breaks is bound poorly by the protein. Once formed, the DNA-protein complex is stable, resisting dissociation in high salt. However, when a DNA strand is broken, the complex appears to dissociate. The protein binds equally well to form I DNA of phi X174 or the plasmid pBR322, but has a higher affinity for a hybrid plasmid containing a cloned region of Drosophila melanogaster satellite DNA.     

Purification of Escherichia coli DNA photolyase

Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction. We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G. B., Smith, F. W., and Sancar, A. (1983) Nucleic Acids Res. 11, 6667-6678). Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme. The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions. The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light. The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E. coli.     

Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.     

Interaction of restriction and modification enzyme EcoRII with synthetic DNA fragments. VII. The study of complex-formation of endonuclease EcoRII with substrates containing natural and modified recognition sites

As shown by a nitrocellulose filter binding assay, in the absence of Mg2+ EcoRII restriction endonuclease binds specifically to a set of synthetic concatemer DNA duplexes of varying chain length, containing natural and modified recognition sites of this enzyme. The binding of the substrates with the central AT, TT or AA-pair in the recognition site decreases at AT greater than TT much greater than AA. Substitution of the pyrophosphate bond at the cleavage site for the phosphodiester or phosphoramide bond produces little influence on the stability of the complexes. The affinity of the enzyme for nonspecific sites is two orders of magnitude less than that for the specific EcoRII sequences. Equilibrium association constant for a substrate with one recognition site is 3.9 X 10(8) M-1. Addition of Mg2+ leads to the destabilization of the EcoRII endonuclease complex with DNA duplex, containing pyrophosphate bonds. The dissociation rate constants and the lifetime of the EcoRII endonuclease--synthetic substrates complexes have been determined.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

Cloning of the phr gene and amplification of photolyase in Escherichia coli.

A new technique is developed for physically enriching recombinant DNA molecules in an in vitro recombination mixture. UV-irradiation of the donor DNA before recombination enables photoreactivating enzyme (PRE) (deoxyribodipyrimidine photolyase, EC 4.1.99.3) to attach to the donor segments in recombinant molecules. This attached protein causes retention of the recombinant molecules on a nitrocellulose filter, while molecules not containing donor DNA pass through. The bound DNA is repaired of its UV damage and released for insertion into cells by exposure to photoreactivating light in situ, yielding approx. 350-fold enrichment. Although applicable to any gene, this procedure has been used in cloning the Escherichia coli phr gene itself, permitting 100-fold amplification of the gene product in vivo.     

Purification and properties of Methanobacterium thermoautotrophicum DNA photolyase

We have purified DNA photolyase from the autotrophic anaerobic archaebacterium Methanobacterium thermoautotrophicum to near homogeneity by a two-column affinity chromatography. The purified enzyme has an Mr = 60,000 and shows near UV absorption peak at 440 nm and a fluorescence emission maximum at 462 nm indicating that it contains 8-hydroxy-5-deazaflavin (coenzyme F420) as an intrinsic chromophore. The photolyase binds with high specificity to thymine dimer in DNA with an equilibrium binding constant, KA = 1.4 x 10(9) M-1, and a dissociation rate constant, koff = 1.4 x 10(-4) s-1 (t1/2 = 43 min). Despite 6-fold higher affinity compared to the folate-containing Escherichia coli photolyase the two enzymes apparently contact the same phosphates around the thymine dimer: the phosphate immediately 5' and the three phosphates immediately 3' to the dimer on the damaged strand and the phosphate across from the dimer in the minor groove on the complementary strand. The absolute action spectrum of the Methanobacterium photolyase in the 400-500-nm region closely matches the absorption of the enzyme-bound F420. The quantum yield (phi) over this region is constant and is approximately 0.2. The value is measurably smaller than the quantum yields reported for other DNA photolyases.     

DNA photolyase of enterococci: possible explanation for its low sunlight inactivation rate

DNA photolyase is perhaps the most ancient and direct arsenal in curing the UV-induced dimers formed in the microbial genome. Out of two cofactors of the enzyme, catalytic and light harvesting, differences in the latter have provided basis for categorizing photolyases of prokaryotes as folate and deazaflavin types. In the present study, the homology modeling of DNA photolyase of Enterococcus faecalis was undertaken. The predicted models were structurally compared with the crystal structure coordinates of photolyases from Escherichia coli (folate type) and Anacystis nidulans (deazaflavin type). Discrepancies present in the multiple sequence alignment and tertiary structures, particularly at the light harvesting cofactor (methenyltetrahydrofolic acid, MTHF; 8-hydroxy-5-deazaflavin, 8-HDF) binding sites indicated the mechanistic nature of enterococcal photolyase. Concisely, despite the greater holistic homology with folate-type photolyase, enterococcal photolyase was characterized as deazaflavin-type. The presence of 8-HDF binding sites and groove architecture of substrate binding sites were also found supportive in this regard. The inter cofactor distance and/or orientation also implied to the efficient energy transfer in photolyase of Enterococcus in comparison with E. coli. In addition, we observed relatively high protein deformability in the enterococcal genome, which may favors the repair action of photolyase. The findings are expected to provide molecular insights into the difference in sunlight inactivation rate of two important fecal contamination indicators, namely Enterococcus and E. coli.     

Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.     

Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin.

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.     

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.