Towards elucidation of the mechanism of UV1C, a deoxyribozyme with photolyase activity

Among the unexpected chemistries that can be catalyzed by nucleic acid enzymes is photochemistry. We have reported the in vitro selection of a small, cofactor-independent deoxyribozyme, UV1C, capable of repairing thymine dimers in a DNA substrate, most optimally with light at a wavelength of >300 nm. We hypothesized that a guanine quadruplex functioned both as a light antenna and an electron source for the repair of the substrate within the enzyme-substrate complex. Here, we report structural and mechanistic investigations of that hypothesis. Contact-crosslinking and guanosine to inosine mutational studies reveal that the thymine dimer and the guanine quadruplex are positioned close to each other in the deoxyribozyme-substrate complex, and permit us to refine the structure and topology of the folded deoxyribozyme. In exploring the substrate utilization capabilities of UV1C, we find it to be able to repair uracil dimers as well as thymine dimers, as long as they are present in an overall deoxyribonucleotide milieu. Some surprising similarities with bacterial CPD photolyase enzymes are noted.     

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.     

DNA repair mechanism by photolyase: electron transfer path from the photolyase catalytic cofactor FADH(-) to DNA thymine dimer

Photolyase is an enzyme that catalyses photorepair of thymine dimers in UV damaged DNA by electron transfer reaction. The structure of the photolyase/DNA complex is unknown at present. Using crystal structure coordinates of the substrate-free enzyme from E. coli, we have recently built a computer molecular model of a thymine dimer docked to photolyase catalytic site and studied molecular dynamics of the system. In this paper, we present analysis of the electronic coupling and electron transfer pathway between the catalytic cofactor FADH(-) and the pyrimidine dimer by the method of interatomic tunneling currents. Electronic structure is treated in the extended Hückel approximation. The root mean square transfer matrix element is about 6 cm(-1), which is consistent with the experimentally determined rate of transfer. We find that electron transfer mechanism responsible for the repair utilizes an unusual folded conformation of FADH(-) in photolyases, in which the isoalloxazine ring of the flavin and the adenine are in close proximity, and the peculiar features of the docked orientation of the dimer. The tunneling currents show explicitly that despite of the close proximity between the donor and acceptor complexes, the electron transfer mechanism between the flavin and the thymine bases is not direct, but indirect, with the adenine acting as an intermediate. These calculations confirm the previously made conclusion based on an indirect evidence for such mechanism.     

Effects of temperature on the photoreactivation of ultraviolet-b–induced dna damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6-4 photoproducts) induced by ultraviolet-B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet-A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6-4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6-4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6-4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6-4 photoproducts. Significant repair of both thymine dimers and 6-4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6-4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.     

Effect of serotonin on the yield of UV- and x ray-induced DNA damage

Using two-dimensional thin-layer chromatography, the effect of serotonin on the yield of thymine dimers and on cleavage of the N-glycosidic bond in the DNA irradiated with ultraviolet (UV) light and X-ray was studied. Bound serotonin was shown to reduce the synthesis of UV-induced thymine dimers but had no effect on the number of X-ray-induced breaks in the N-glycoside bonds in thymidine residues. The data obtained are discussed in terms of the mechanisms of serotonin involvement in the photoprotection of yeast cells from the lethal action of UV and X-ray irradiations.     

Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.     

Purified scrapie prions resist inactivation by UV irradiation.

The development of effective purification protocols has permitted evaluation of the resistance of isolated scrapie prions to inactivation by UV irradiation at 254 nm. Prions were irradiated on ice with doses of UV light ranging up to 120,000 J/m2. UV dosimetry experiments, performed with Saccharomyces cerevisiae plasmid DNA or eucaryotic cells, indicated that under these experimental conditions an incident UV dose of 10 J/m2 formed 2 thymine dimers per 5.1 X 10(6) daltons of eucaryotic cell DNA. The D37 values for scrapie prions ranged from 17,000 to 22,000 J/m2; D37 values were also determined for virus, viroid, and enzyme controls. The number of pyrimidine dimers formed was correlated with the D37 values obtained for irradiated prions and target nucleic acids. The D37 value for bacteriophage M13, 6.5 J/m2, occurred at a dose that would form 0.56 dimers per target genome; the D37 for potato spindle tuber viroid, 4,800 J/m2, occurred at a dose that would form about 24 dimers per target viroid. The D37 value for an EcoRI restriction site, a target of 12 bases, occurred at a dose that would correspond to the formation of 0.89 thymine dimers per target site. The D37 value for prions occurred at a dose that would form 1 dimer in every 4 bases of single-stranded target nucleic acid. If the putative scrapie nucleic acid were double-stranded and readily repairable after UV damage, then the prion D37 value could reflect a nucleic acid molecule of 30 to 45 base pairs. While the D37 value for prions fell within the range of pure protein targets, our experiments cannot eliminate the possibility that a prion contains a small, highly protected nucleic acid molecule.     

Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts

We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone. The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV. Furthermore, the treatment of UV-irradiated DNA with photolyase from E. coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA. A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts. These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA. Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells. Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.     

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.     

Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.     

Identification and characterization of a prokaryotic 6-4 photolyase from Synechococcus elongatus with a deazariboflavin antenna chromophore

Synechococcus elongatus, formerly known as Anacystis nidulans, is a representative species of cyanobacteria. It is also a model organism for the study of photoreactivation, which can be fully photoreactivated even after receiving high UV doses. However, for a long time, only one photolyase was found in S. elongatus that is only able to photorepair UV induced cyclobutane pyrimidine dimers (CPDs) in DNA. Here, we characterize another photolyase in S. elongatus, which belongs to iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP), a subtype of prokaryotic 6–4 photolyases. This photolyase was named SePhrB that could efficiently photorepair 6–4 photoproducts in DNA. Chemical analyses revealed that SePhrB contains a catalytic FAD cofactor and an iron-sulfur cluster. All of previously reported FeS-BCPs contain 6,7-dimethyl-8-ribityllumazine (DMRL) as their antenna chromophores. Here, we first demonstrated that SePhrB possesses 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF) as an antenna chromophore. Nevertheless, SePhrB could be photoreduced without external electron donors. After being photoreduced, the reduced FAD cofactor in SePhrB was extremely stable against air oxidation. These results suggest that FeS-BCPs are more diverse than expected which deserve further investigation.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Molecular and functional analysis of the (6-4) photolyase from the hexactinellid Aphrocallistes vastus

The hexactinellid sponges (phylum Porifera) represent the phylogenetically oldest metazoans that evolved 570-750 million years ago. At this period exposure to ultraviolet (UV) light exceeded that of today and it may be assumed that this old taxon has developed a specific protection system against UV-caused DNA damage. A cDNA was isolated from the hexactinellid Aphrocallistes vastus which comprises high sequence similarity to genes encoding the protostomian and deuterostomian (6-4) photolyases. Subsequently functional studies were performed. It could be shown that the sponge gene, after transfection into mutated Escherichia coli, causes resistance of the bacteria against UV light. Recombinant sponge photolyase was prepared to demonstrate that this protein binds to DNA treated with UV light (causing the formation of thymine dimers). Finally, it is shown that the photolyase gene is strongly expressed in the upper part of the animals and not in their middle part or their base. It is concluded that sponges not only have an excision DNA repair system, as has been described earlier by us, but also a photolyase-based photo-reactivating system.     

Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase

More than 50 years ago, initial experiments on enzymatic photorepair of ultraviolet (UV)-damaged DNA were reported [Proc. Natl. Acad. Sci. U. S. A. 35 (1949) 73]. Soon after this discovery, it was recognized that one enzyme, photolyase, is able to repair UV-induced DNA lesions by effectively reversing their formation using blue light. The enzymatic process named DNA photoreactivation depends on a non-covalently bound cofactor, flavin adenine dinucleotide (FAD). Flavins are ubiquitous redox-active catalysts in one- and two-electron transfer reactions of numerous biological processes. However, in the case of photolyase, not only the ground-state redox properties of the FAD cofactor are exploited but also, and perhaps more importantly, its excited-state properties. In the catalytically active, fully reduced redox form, the FAD absorbs in the blue and near-UV ranges of visible light. Although there is no direct experimental evidence, it appears generally accepted that starting from the excited singlet state, the chromophore initiates a reductive cleavage of the two major DNA photodamages, cyclobutane pyrimidine dimers and (6-4) photoproducts, by short-distance electron transfer to the DNA lesion. Back electron transfer from the repaired DNA segment is believed to eventually restore the initial redox states of the cofactor and the DNA nucleobases, resulting in an overall reaction with net-zero exchanged electrons. Thus, the entire process represents a true catalytic cycle.Many biochemical and biophysical studies have been carried out to unravel the fundamentals of this unique mode of action. The work has culminated in the elucidation of the three-dimensional structure of the enzyme in 1995 that revealed remarkable details, such as the FAD-cofactor arrangement in an unusual U-shaped configuration. With the crystal structure of the enzyme at hand, research on photolyases did not come to an end but, for good reason, intensified: the geometrical structure of the enzyme alone is not sufficient to fully understand the enzyme's action on UV-damaged DNA. Much effort has therefore been invested to learn more about, for example, the geometry of the enzyme-substrate complex, and the mechanism and pathways of intra-enzyme and enzyme ↔DNA electron transfer. Many of the key results from biochemical and molecular biology characterizations of the enzyme or the enzyme-substrate complex have been summarized in a number of reviews. Complementary to these articles, this review focuses on recent biophysical studies of photoreactivation comprising work performed from the early 1990s until the present.     

Absolute action spectrum of E-FADH2 and E-FADH2-MTHF forms of Escherichia coli DNA photolyase

Escherichia coli DNA photolyase mediates photorepair of pyrimidine dimers occurring in UV-damaged DNA. The enzyme contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolylpolyglutamate (MTHF). To define the roles of the two chromophores in the photochemical reaction(s) resulting in DNA repair and the effect of DNA structure on the photocatalytic step, we determined the absolute action spectra of the enzyme containing only FADH2 (E-FADH2) or both chromophores (E-FADH2-MTHF), with double- and single-stranded substrates and with substrates of different sequences in the immediate vicinity of the thymine dimer. We found that the shape of the action spectrum of E-FADH2 matches that of the absorption spectrum with a quantum yield phi (FADH2) = 0.69. The action spectrum of E-FADH2-MTHF is also in a fairly good agreement with the absorption spectrum with phi (FADH2-MTHF) = 0.59. From these values and from the previously established properties of the two chromophores, we propose that MTHF transfers energy to FADH2 with a quantum yield of phi epsilon T = 0.8 and that 1FADH2 singlet transfers an electron to or from the dimer with a quantum yield phi ET = 0.69. The chemical nature of the chromophores did not change after several catalytic cycles. The enzyme repaired a thymine dimer in five different sequence contexts with the same efficiency. Similarly, single- and double-stranded DNAs were repaired with the same overall quantum yield.     

UV-B-Inducible and Temperature-Sensitive Photoreactivation of Cyclobutane Pyrimidine Dimers in Arabidopsis thaliana

Removal of cyclobutane pyrimidine dimers (CBPDs) in vivo from the DNA of UV-irradiated eight-leaf seedlings of Arabidopsis thaliana was rapid in the presence of visible light (half-life about 1 hour); removal of CBPDs in the dark, presumably via excision repair, was an order of magnitude slower. Extracts of plants contained significant photolyase in vitro, as assayed by restoration of transforming activity to UV-irradiated Escherichia coli plasmids; activity was maximal from four-leaf to 12-leaf stages. UV-B treatment of seedlings for 6 hours increased photolyase specific activity in extracts twofold. Arabidopsis photolyase was markedly temperature-sensitive, both in vitro (half-life at 30°C about 12 minutes) and in vivo (half-life at 30°C, 30 to 45 minutes). The wavelength dependency of the photoreactivation cross-section showed a broad peak at 375 to 400 nm, and is thus similar to that for maize pollen; it overlaps bacterial and yeast photolyase action spectra.     

Biochemical and biological properties of DNA photolyases derived from utraviolet-sensitive rice cultivars

Class I and class II CPD photolyases are enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We identified three amino acid changes, Gln126Arg, Gly255Ser, and Gln296His, among which Gln but not His at 296 is important for complementing phr-defective E. coli, binding UV-damage in E. coli, and binding thymine dimers in vitro. The photolyase with Gln at 296 has an apoenzyme:FAD ratio of 1 : 0.5 and that with His at 296 has an apoenzyme:FAD ratio of 1 : 0.12-0.25, showing a role for Gln at 296 in the binding of FAD not in the binding of thymine dimer. Concerning Gln or Arg at 126, the biochemical activity of the photolyases purified from E. coli and complementing activity for phr-defective E. coli are similarly proficient. However, the sensitivity to UV of cultivars differs depending on whether Gln or Arg is at 126. The role of Gln and Arg at 126 for photoreactivation in rice is discussed.     

Cis-syn thymidine dimer repair by DNA photolyase in real time

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.     

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.