Enzymatic photoreactivation: 50 years and counting.

The discovery of enzymatic photoreactivation and of photolyase produced a paradigm shift in the way investigators thought about the cellular consequences of DNA damage and about how these consequences could be avoided. The in vitro photoreactivation system, which utilized crude extracts from Saccharomyces cerevisiae as the source of photolyase, not only provided information about the mechanism of photoreactivation, but also played an important role in the discovery of nucleotide excision repair (NER) and the identification of the pyrimidine dimer as the primary lethal lesion induced by 254 nm radiation. More recently, mechanistic studies using homogenous purified yeast photolyase have yielded insight into how DNA repair enzymes recognize specific structures in DNA, while investigations looking at the repair of lesions in chromatin have begun to elucidate how DNA repair enzymes deal with damage in the context of eukaryotic chromosomes. Additionally, genetic and molecular studies of PHR1, the S. cerevisiae gene encoding the apoenzyme of photolyase, have led to the identification of previously unknown damage-responsive transcriptional regulators.

RPH1 and GIS1 are damage-responsive repressors of PHR1.

The Saccharomyces cerevisiae DNA repair gene PHR1 encodes a photolyase that catalyzes the light-dependent repair of pyrimidine dimers. PHR1 expression is induced at the level of transcription by a variety of DNA-damaging agents. The primary regulator of the PHR1 damage response is a 39-bp sequence called URS(PHR1) which is the binding site for a protein(s) that constitutes the damage-responsive repressor PRP. In this communication, we report the identification of two proteins, Rph1p and Gis1p, that regulate PHR1 expression through URS(PHR1). Both proteins contain two putative zinc fingers that are identical throughout the DNA binding region, and deletion of both RPH1 and GIS1 is required to fully derepress PHR1 in the absence of damage. Derepression of PHR1 increases the rate and extent of photoreactivation in vivo, demonstrating that the damage response of PHR1 enhances cellular repair capacity. In vitro footprinting and binding competition studies indicate that the sequence AG(4) (C(4)T) within URS(PHR1) is the binding site for Rph1p and Gis1p and suggests that at least one additional DNA binding component is present in the PRP complex.

Evidence for dinucleotide flipping by DNA photolyase.

DNA photolyases repair pyrimidine dimers via a reaction in which light energy drives electron donation from a catalytic chromophore, FADH-, to the dimer. The crystal structure of Escherichia coli photolyase suggested that the pyrimidine dimer is flipped out of the DNA helix and into a cavity that leads from the surface of the enzyme to FADH-. We have tested this model using the Saccharomyces cerevisiae Phr1 photolyase which is >50% identical to E. coli photolyase over the region comprising the DNA binding domain. By using the bacterial photolyase as a starting point, we modeled the region encompassing amino acids 383-530 of the yeast enzyme. The model retained the cavity leading to FADH- as well as the band of positive electrostatic potential which defines the DNA binding surface. We found that alanine substitution mutations at sites within the cavity reduced both substrate binding and discrimination, providing direct support for the dinucleotide flip model. The roles of three residues predicted to interact with DNA flanking the dimer were also tested. Arg452 was found to be particularly critical to substrate binding, discrimination, and photolysis, suggesting a role in establishing or maintaining the dimer in the flipped state. A structural model for photolyase-dimer interaction is presented.

Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair. One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase. Basal-level and damage-induced expression of PHR1 require an upstream activation sequence, UAS(PHR1), which has homology with DRC elements found upstream of at least 19 other DNA repair and DNA metabolism genes in yeast. Here we report the identification of the UME6 gene of S. cerevisiae as a regulator of UAS(PHR1) activity. Multiple copies of UME6 stimulate expression from UAS(PHR1) and the intact PHR1 gene. Surprisingly, the effect of deletion of UME6 is growth phase dependent. In wild-type cells PHR1 is induced in late exponential phase, concomitant with the initiation of glycogen accumulation that precedes the diauxic shift. Deletion of UME6 abolishes this induction, decreases the steady-state concentration of photolyase molecules and PHR1 mRNA, and increases the UV sensitivity of a rad2 mutant. Despite the fact that UAS(PHR1) does not contain the URS1 sequence, which has been previously implicated in UME6-mediated transcriptional regulation, we find that Ume6p binds to UAS(PHR1) with an affinity and a specificity similar to those seen for a URS1 site. Similar binding is also seen for DRC elements from RAD2, RAD7, and RAD53, suggesting that UME6 contributes to the regulated expression of a subset of damage-responsive genes in yeast.

Promoter elements of the PHR1 gene of Saccharomyces cerevisiae and their roles in the response to DNA damage.

The PHR1 gene of Saccharomyces cerevisiae encodes the apoenzyme for the DNA repair enzyme photolyase. PHR1 transcription is induced in response to 254 nm radiation and a variety of chemical damaging agents. We report here the identification of promoter elements required for PHR1 expression. Transcription is regulated primarily through three sequence elements clustered within a 120 bp region immediately upstream of the translational start site. A 20 bp interrupted palindrome comprises UASPHR1 and is responsible for 80-90% of basal and induced expression. UASPHR1 alone can activate transcription of a CYC1 minimal promoter but does not confer damage responsiveness. In the intact PHR1 promoter UAS function is dependent upon an upstream essential sequence (UES). URSPHR1 contains a binding site for the damage-responsive repressor Prp; consistent with this role, deletion or specific mutations of the URS increase basal level expression and decrease the induction ratio. Deletion of URSPHR1 also eliminates the requirement for UESPHR1 for promoter activation, indicating that the UES attenuates Prp-mediated repression. Sequences within UASPHR1 are similar to regulatory sequences found upstream of both damage responsive and nonresponsive genes involved in DNA repair and metabolism.

The role of conserved amino acids in substrate binding and discrimination by photolyase.

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. We have utilized chemical modification and site-directed mutagenesis to probe the interactions involved in substrate recognition by the yeast photolyase Phr1. Lys517 was protected from reductive methylation in the presence of substrate, but not in its absence, and the specific and nonspecific association constants for substrate binding by Phr1 (Lys517-->Ala) were decreased 10-fold. These results establish a role for Lys517 in substrate binding. Mutations at Arg507, Lys463, and Trp387 reduced both the overall affinity for substrate and substrate discrimination. Sites of altered interactions in ES complexes were identified by methylation and ethylation interference techniques. Interaction with the base immediately 3' to the dimer was altered in the Phr1(Lys517-->Ala). DNA complex, whereas interactions with the phosphate and base immediately 5' to the dimer were reduced when Phr1(Arg507-->Ala) bound substrate. Multiple interactions 5' and 3' to the dimer were perturbed in complexes containing Phr1(Trp387-->Ala) or Phr1(Lys463-->Ala). In addition the quantum yield for dimer photolysis by Phr1(Trp387-->Ala) was reduced 3-fold. The locations of these mutations establish that a portion of the DNA binding domain is comprised of residues in the highly conserved carboxyl-terminal half of the enzyme.

Identification of chromophore binding domains of yeast DNA photolyase.

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription.

DNA photolyases: physical properties, action mechanism, and roles in dark repair.

DNA photolyases catalyze the light-dependent repair of cis,syn-cyclobutane dipyrimidines (pyrimidine dimers). Although the phenomenon of enzymatic photoreactivation was first described 40 years ago and photolyases were the first enzymes shown unequivocally to effect DNA repair, it has only been in the last 8 years that sufficient quantities of the enzymes have been purified to permit detailed studies of their physical properties, identification of their intrinsic chromophores, and elucidation of the mechanisms of dimer recognition and photolysis. In addition several of the genes encoding these enzymes have now been cloned and sequenced. These studies have revealed remarkable functional and structural conservation among these evolutionarily ancient enzymes and have identified a new role for photolyases in dark-repair processes which has implications for the mechanism of nucleotide excision repair in both prokaryotes and eukaryotes.

Expression of the yeast PHR1 gene is induced by DNA-damaging agents.

The PHR1 gene of Saccharomyces cerevisiae encodes a photolyase which repairs specifically and exclusively pyrimidine dimers, the most frequent lesions induced in DNA by far-UV radiation. We have asked whether expression of PHR1 is modulated in response to UV-induced DNA damage and to DNA-damaging agents that induce lesions structurally dissimilar to pyrimidine dimers. Using a PHR1-lacZ fusion gene in which expression of beta-galactosidase is regulated by PHR1 5' regulatory elements, we found that exposure of cells to 254-nm light, 4-nitroquinoline-N-oxide, methyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine induced synthesis of increased amounts of fusion protein. In contrast to these DNA-damaging agents, neither heat shock nor exposure to photoreactivating light elicited a response. Induction by far-UV radiation was evident both when the fusion gene was carried on a multicopy plasmid and when it replaced the endogenous chromosomal copy of PHR1, and it was accompanied by an increase in the steady-state concentration of PHR1-lacZ mRNA. Northern (RNA) blot analysis of PHR1 mRNA encoded by the chromosomal locus was consistent with either enhanced transcription of PHR1 after DNA damage or stabilization of the transcripts. Neither the intact PHR1 or RAD2 gene was required for induction. Comparison of the region of PHR1 implicated in regulation of its expression with other damage-inducible genes from yeast cells revealed a common conserved sequence that is present in the PHR1, RAD2, and RNR2 genes and is required for damage inducibility of the latter two genes. These sequences may constitute elements of a damage-responsive regulon in S. cerevisiae.

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.

Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.

Construction of plasmids which lead to overproduction of yeast PHR1 photolyase in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase which is normally present in fewer than 300 copies per cell. We have constructed plasmids in which PHR1 expression in yeast and Escherichia coli is under the control of strong, inducible promoters thereby leading to the regulated overproduction of biologically active photolyase. Under inducing conditions, E. coli cells carrying the tac-PHR1 plasmid pCB1241 accumulate up to 8% of total cellular protein as yeast photolyase; similarly, the GAL10-PHR1 fusion plasmid pGBS107 directs the synthesis of at least 1800-2400 molecules of photolyase per log-phase yeast cell. In both plasmids translation begins at the first ATG in the PHR1 open reading frame (ORF). Constructs in which translation initiates at the second or third ATG fail to complement yeast and E. coli phr1 mutations, indicating that the first ATG in the PHR1 ORF is the translational start site in vivo and that all or part of the N-terminal 78 amino acids are required for activity.

Identification of the second chromophore of Escherichia coli and yeast DNA photolyases as 5,10-methenyltetrahydrofolate.

Denaturation of DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) from Escherichia coli with guanidine hydrochloride or acidification to pH 2 released, in addition to FAD, a chromophore with the spectral and chromatographic properties of a reduced pterin. Treatment of the enzyme with iodine prior to acidification converted the chromophore to a stable, oxidized derivative, which was resolved by HPLC into four species with identical spectral properties. The same species, in the same distribution, were obtained from the yeast enzyme. The material isolated from the iodine-oxidized enzyme was shown to be a pterin by conversion to pterin-6-carboxylic acid with alkaline permanganate and was found to release glutamate upon acid hydrolysis. The presence of 10-formylfolate in the isolated, oxidized chromophore was demonstrated by absorption and fluorescence spectroscopy and by deformylation and conversion to folic acid. Analysis of the distribution of polyglutamates revealed that the four species identified by HPLC corresponded to the tri-, tetra-, penta-, and hexaglutamate derivatives of 10-formylfolate. The results were consistent with gamma linkages in the triglutamate derivative with additional glutamates linked via the alpha-carboxyl group of the preceding residue. Treatment with rat plasma hydrolase produced the monoglutamate derivative of 10-formylfolate. The native, enzyme-bound form of the folate cofactor was identified as 5,10-methenyltetrahydrofolylpolyglutamate by effecting release and isolation at low pH to protect the 5,10-methenyl bridge and preserve the reduced pyrazine ring structure.

Purification of the yeast PHR1 photolyase from an Escherichia coli overproducing strain and characterization of the intrinsic chromophores of the enzyme.

We have placed the PHR1 gene of Saccharomyces cerevisiae under the transcriptional and translational control of the tac expression cartridge. Under inducing conditions Escherichia coli cells harboring plasmids carrying this construct accumulate approximately 8% of total cellular protein as the Phr1 photolyase. Using a strain devoid of E. coli photolyase activity, we have obtained milligram quantities of the yeast enzyme at greater than 95% purity and have characterized the enzyme. Phr1 photolyase is a monomer in solution with an Mr of 60,000, has a turnover number of 0.7 dimers min-1 molecule-1 in vitro, exhibits absorbance maxima at lambda = 277 and 377 nm, and has a fluorescence excitation maximum at 390 nm and an emission maximum at 475 nm. The near UV absorbance peak is shown to reflect the contributions of two intrinsic chromophores which are noncovalently bound to the enzyme. Spectroscopic, fluorescence, and thin layer chromatographic studies indicate that one of these chromophores is 1,5-reduced FAD rather than 4a,5-reduced FAD as previously proposed (Iwatsuki, N., Joe, C. O., and Werbin, H. (1980) Biochemistry 19, 1172-1176), while the other chromophore has properties similar to the second chromophore of E. coli photolyase. The fact that yeast and E. coli photolyases are similar both with respect to amino acid sequence and chromophore composition provides strong evidence that the enzymes share a common action mechanism which may also be utilized by photolyases from other organisms throughout the phylogenetic tree.

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.

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.

Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis.

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.