Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase

The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location.     

Role of His-16 in turnover of T4 pyrimidine dimer glycosylase

Previously, the histidine residue at position 16 in the mature T4 pyrimidine dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle.     

Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA

The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.     

Uncoupling of nucleotide flipping and DNA bending by the t4 pyrimidine dimer DNA glycosylase

Bacteriophage T4 pyrimidine dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane pyrimidine dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5'-thymine of a thymine-thymine (TT) dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60 degrees in the enzyme-substrate (ES) complex. To examine the kinetic details of the precatalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately assess nucleotide flipping and DNA bending. The fluorescent adenine base analogue, 2-aminopurine (2-AP), placed opposite an abasic site analogue, tetrahydrofuran, exhibited a 2.8-fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping, kon and koff pre-steady-state kinetic measurements were determined. DNA bending was assessed by fluorescence resonance energy transfer using fluorescent donor-acceptor pairs located at the 5'-ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5-fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.     

Properties of a UV-sensitive Neurospora strain defective in pyrimidine dimer excision

The UV-sensitive Neurospora strain uvs-2 is known to resemble the excision-defective uvr mutants of E. coli K12 in being both excision-defective and highly UV mutable. As shown in this report, the uvs-2 strain also resembles the uvr mutants in its ability to remain photoreactivable when held in the dark for 2 h between UV-irradiation and photoreactivating light exposure, and in its maintenance of the same spontaneous deletion rate as wild type strains.Unlike the E. coli uvr mutants, however, this strain is sensitive to ionizing radiation and shows an increase in survival when held for 2 h in distilled water before plating (liquid-holding recovery [LHR]). The strain is three times more sensitive to X-rays than the wild type strain. It is also sensitive to nitrosoguanidine (MNNG). Sensitivity to UV, X-rays and MNNG appears to be under the control of a single gene.These properties suggest that the repair defect in the Neurospora uvs-2 mutant is different from those of the uvr mutants of E. coli K12.     

Effects of DNA looping on pyrimidine dimer formation.

We have assessed the effects of DNA curvature on pyrimidine dimer (PD) formation by examining the pattern of PD formation in DNA held in a loop by lambda repressor. The loop region was composed of diverse DNA sequences such that potential PD sites occurred throughout the loop. PD formation in the loop occurred with peaks at approximately 10 base intervals, just 3' of where the bending of the DNA was inferred to be toward the major groove. This relationship between the peaks and the DNA curvature is essentially identical to that observed in the nucleosome. This indicates that DNA curvature is the major source of the periodicity of PD formation in the nucleosome, and supports an earlier model of the conformation of nucleosomal DNA based on PD formation. DNA loops containing diverse sequences should be of general value for assessing the effects of DNA curvature on DNA modification by other agents used to probe DNA-protein interactions and DNA conformation.     

Light-induced activation of class II cyclobutane pyrimidine dimer photolyases

Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6–4) photolyase. By ENDOR spectroscopy, the local environment of the flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FAD^ox, have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADH from FAD^ox, and subsequently FADH^? from FADH, proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution.     

The catalytic mechanism of a pyrimidine dimer-specific glycosylase (pdg)/abasic lyase, Chlorella virus-pdg

The repair of UV light-induced cyclobutane pyrimidine dimers can proceed via the base excision repair pathway, in which the initial step is catalyzed by DNA glycosylase/abasic (AP) lyases. The prototypical enzyme studied for this pathway is endonuclease V from the bacteriophage T4 (T4 bacteriophage pyrimidine dimer glycosylase (T4-pdg)). The first homologue for T4-pdg has been found in a strain of Chlorella virus (strain Paramecium bursaria Chlorella virus-1), which contains a gene that predicts an amino acid sequence homology of 41% with T4-pdg. Because both the structure and critical catalytic residues are known for T4-pdg, homology modeling of the Chlorella virus pyrimidine dimer glycosylase (cv-pdg) predicted that a conserved glutamic acid residue (Glu-23) would be important for catalysis at pyrimidine dimers and abasic sites. Site-directed mutations were constructed at Glu-23 to assess the necessity of a negatively charged residue at that position (Gln-23) and the importance of the length of the negatively charged side chain (Asp-23). E23Q lost glycosylase activity completely but retained low levels of AP lyase activity. In contrast, E23D retained near wild type glycosylase and AP lyase activities on cis-syn dimers but completely lost its activity on the trans-syn II dimer, which is very efficiently cleaved by the wild type cv-pdg. As has been shown for other glyscosylases, the wild type cv-pdg catalyzes the cleavage at dimers or AP sites via formation of an imino intermediate, as evidenced by the ability of the enzyme to be covalently trapped on substrate DNA when the reactions are carried out in the presence of a strong reducing agent; in contrast, E23D was very poorly trapped on cis-syn dimers but was readily trapped on DNA containing AP sites. It is proposed that Glu-23 protonates the sugar ring, so that the imino intermediate can be formed.     

Influence of puromycin aminonucleoside on photoreactivation and pyrimidine dimer repair

We have measured the effect of puromycin aminonucleoside (PAN) on photoreactivation of mutations and loss of pyrimidine dimers in structural gene Strep A in ultraviolet (UV)-irradiatedEscherichia coli B/r T-cells. Photoreactivating illumination between 3100 and 4000 Å wavelengths for 45 min on brain-heart infusion (BHI) of B/r T-eliminated 55% of the pyrimidine dimers and 75% of the mutations in gene Strep A. When BHI-PAN medium was employed, there was a 45% loss of dimers and 73% reduction in mutations. Incubation for 3.5 on BHI or BHI-PAN prior to such treatment results in no loss of mutations. These results suggest that the photoreactivity of mutations in gene Strep A is related to the ability of the cells to repair pyrimidine dimers. However, no direct correlation has been made between the observed repair of mutations and repair of pyrimidine dimers. Further experiments on the kinetics of the repair process, designed to elucidate the mechanism of PAN action, show that slope increases with increasing concentration of the drug that follows an enzyme-like pattern.     

The native cyclobutane pyrimidine dimer photolyase of rice is phosphorylated

The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containing DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54- and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia coli was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54- and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chromophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. coli-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. coli-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice.     

Efficiency of pyrimidine dimer formation in Escherichia coli across UV wavelengths

AIMS: Inactivation of Escherichia coli as a function of ultraviolet (UV) wavelength was investigated by using the endonuclease-sensitive site (ESS) assay to quantify pyrimidine dimer formation. METHODS AND RESULTS: Ultraviolet dose-response curves were determined based on both log reduction in colony-forming units (CFU) and endonuclease-sensitive sites per kb DNA (ESS/kb) for monochromatic 254-nm low-pressure (LP) UV, polychromatic medium-pressure (MP) UV, 228 and 289-nm UV irradiation. UV irradiation from LP and MP UV sources were approx. equal in both CFU reduction and pyrimidine dimer formation at all UV doses studied; 228-nm irradiation was less effective than LP or MP, and 289-nm irradiation was the least effective in both CFU reduction and pyrimidine dimer formation. These results are in qualitative agreement with the absorption spectrum of pyrimidine bases in DNA. Results indicated an approx. linear relationship between ESS/kb and log CFU reduction. CONCLUSIONS: Formation of pyrimidine dimers in genomic DNA is primarily responsible for UV inactivation of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributed to fundamental understanding of UV disinfection and aids in UV reactor design.     

Repair of pyrimidine dimer ultraviolet light photoproducts by human cell extracts

A newly developed method allows human cell extracts to carry out repair synthesis on ultraviolet light irradiated closed circular plasmid DNA [Wood, R. D., Robins, P., & Lindahl, T. (1988) Cell 53, 97-106]. The identity of the photodamage that leads to this repair replication was investigated. Removal of stable pyrimidine hydrates from irradiated plasmid pAT153 did not significantly affect the amount of repair replication in the fluence range of 0-450 J/m2, because of the low yield of these products and their short DNA repair patch size. Photoreactivation of irradiated DNA using purified Escherichia coli DNA photolyase to remove more than 95% of the cyclobutane dimers from the DNA reduced the observed repair synthesis by 20-40%. The greater part of the repair synthesis is highly likely to be caused by (6-4) pyrimidine dimer photoproducts. This class of lesions is rapidly repaired by mammalian cells, and their removal is known to be important for cell survival after ultraviolet irradiation.     

Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase.

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.     

Rotational position of a 5-methylcytosine-containing cyclobutane pyrimidine dimer in a nucleosome greatly affects its deamination rate

C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations are proposed to arise from the insertion of A by DNA polymerase η opposite the T that results from deamination of the methylC ((m)C) within the CPD. Although the frequency of CPD formation and repair is modestly modulated by its rotational position within a nucleosome, the effect of position on the rate of (m)C deamination in a CPD has not been previously studied. We now report that deamination of a T(m)C CPD whose sugar phosphate backbone is positioned against the histone core surface decreases by a factor of 4.7, whereas that of a T(m)C CPD positioned away from the surface increases by a factor of 8.9 when compared with unbound DNA. Because the (m)Cs undergoing deamination are in similar steric environments, the difference in rate appears to be a consequence of a difference in the flexibility and compression of the two sites due to DNA bending. Considering that formation of the CPD positioned away from the surface is also enhanced by a factor of two, a T(m)CG site in this position might be expected to have up to an 84-fold higher probability of resulting in a UV-induced (m)C to T mutation than one positioned against the surface. These results indicate that rotational position may play an important role in the formation of UV-induced C to T mutation hotspots, as well as in the mutagenic mechanism of other DNA lesions.     

den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a DNA glycosylase activity that produced alkali-labile sites in irradiated DNA and an apyrimidinic/apurinic endonuclease activity that converted these sites to nicks. Both activities could be detected in the presence of 10 mM EDTA. In experiments designed to determine which of the activities is coded by the denV gene, we found that the glycosylase was more heat labile in extracts of Escherichia coli infected with either of two thermosensitive denV mutants than in extracts of cells infected with wild-type T4. In contrast, apyrimidinic/apurinic endonuclease activity was no more heat labile in extracts of the former than in extracts of the latter. Our results indicate that the denV gene codes for a DNA glycosylase specific for pyrimidine dimers.     

UV radiation-sensitive norin 1 rice contains defective cyclobutane pyrimidine dimer photolyase

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.     

Determination of pyrimidine dimer unwinding angle by measurement of DNA electrophoretic mobility

The unwinding angle produced by the formation of one pyrimidine dimer has been estimated to be ?14.3 ° ±0.2 °. This value has been obtained by titrating the number of pyrimidine dimers necessary to reduce the number of superhelical turns by one in each topoisomer obtained by treatment of a supercoiled DNA with DNA topoisomerase I.     

DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis-syn-cyclobutane thymine dimer (T[c,s]T). ³¹P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[c,s]T) and d(TAT[c,s]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[c,s]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V_H domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3′-side of the nucleotides onto the H1 and H3 segments, with the 5′-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.