Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1

Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.     

Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide

We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.     

MutY DNA glycosylase: base release and intermediate complex formation

MutY protein, a DNA glycosylase found in Escherichia coli, recognizes dA:dG, dA:8-oxodG, and dA:dC mismatches in duplex DNA, excising the adenine moiety. We have investigated the mechanism of action of MutY, addressing several points of disagreement raised by previous studies of this enzyme. MutY forms a covalent intermediate with its DNA substrate but does not catalyze strand cleavage. The covalent intermediate has a half-life of approximately 2.6 h, 2 orders of magnitude greater than the half-life of Schiff bases formed when E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III react with their respective substrates. The covalent complex between MutY and its DNA substrate involves Lys-142; however, the position of this residue in the presumptive active site differs from that of catalytic residues involved in Schiff base formation associated with endonuclease III and related DNA glycosylases/AP lyases. MutY converts DNA duplexes containing the dA:8-oxodG mispair to a product containing an abasic site; heat-induced cleavage of this product may account for the several reports in the literature that ascribe AP lyase activity to MutY. The MutY-DNA intermediate complex is highly stable and hinders access by Fpg to DNA, thereby avoiding a double-strand break. Cross-linking of MutY to DNA may play an important role in the regulation of base excision repair.     

Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase

An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans. Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol. Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol. The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins. These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein.     

Cloning and characterization of the genes of the CeqI restriction-modification system

Two genes from Corynebacterium equii, a Gram-positive bacterium producing the CeqI restriction-modification enzymes were cloned and sequenced. In vivo restriction experiments, DNA and amino acid sequence data suggest that the two genes code for the endonuclease and the methyltransferase enzymes. However, when the two genes are expressed in E. coli, practically no enzyme activity can be detected in the supernatants of sonicated cells. Based on the DNA sequence data CeqI restriction endonuclease (an EcoRV izoschizomer) consists of 270 amino acid residues with a predicted molecular mass of 31.6 kDa, in good agreement with the previously measured 32 +/- 2 kDa. The methyltransferase is 517 residues long (approx. 60 kDa). The two genes are in opposite orientation and overlap by 37 base pairs on the chromosome. The deduced amino acid sequence of the putative endonuclease gene revealed long stretches of hydrophobic amino acids, that may form the structural basis of the unusual aggregation properties of the restriction endonuclease. The amino acid sequence of the methylase shows homologies with other type II methyltransferases.     

The action of DNA ligase at abasic sites in DNA

Apurinic/apyrimidinic (AP) sites occur frequently in DNA as a result of spontaneous base loss or following removal of a damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5'-deoxyribose phosphate (dRP) residue that must be removed during the base excision repair process. This 5'-dRP group may be removed by AP lyase enzymes that employ a beta-elimination mechanism. This beta-elimination reaction typically involves a transient Schiff base intermediate that can react with sodium borohydride to trap the DNA-enzyme complex. With the use of this assay as well as direct 5'-dRP group release assays, we show that T4 DNA ligase, a representative ATP-dependent DNA ligase, contains AP lyase activity. The AP lyase activity of T4 DNA ligase is inhibited in the presence of ATP, suggesting that the adenylated lysine residue is part of the active site for both the ligase and lyase activities. A model is proposed whereby the AP lyase activity of DNA ligase may contribute to the repair of abasic sites in DNA.     

Cyclobutane pyrimidine dimers in UV-DNA induce release of soluble mediators that activate the human immunodeficiency virus promoter

Ultraviolet (UV) irradiation of human cells induced expression of a stably maintained fusion gene consisting of the human immunodeficiency virus long terminal repeat promoter controlling the bacterial chloramphenicol acetyltransferase gene. Two experiments demonstrated that DNA damage can initiate induction: UV induction was greater in DNA repair-deficient cells from a xeroderma pigmentosum patient than in repair-proficient cells, and transfection of UV-irradiated DNA into unirradiated cells activated gene expression. Increased repair of cyclobutane pyrimidine dimers by T4 endonuclease V abrogated viral gene activation, suggesting that dimers in DNA are one signal leading to increased gene expression. This signal was spread from UV-irradiated cells to unirradiated cells by co-cultivation, implicating the release of soluble factors. Irradiation of cells from DNA repair-deficiency diseases resulted in greater release of soluble factors than irradiation of cells from unaffected individuals. These results suggest that UV-induced cyclobutane pyrimidine dimers can activate the human immunodeficiency virus promoter at least in part by a signal-transduction pathway that includes secretion of soluble mediators.     

Molecular cloning and expression of NlaIII restriction-modification system in E. coli

The NlaIII restriction enzyme isolated from Neisseria lactamica recognizes the sequence 5'-CATG-3', cleaving after the G to generate a four base 3' overhang. The NlaIII methylase and a portion of the NlaIII endonuclease gene were cloned into E. coli by the methylase selection method, and the remaining portion of the NlaIII endonuclease gene was cloned by inverse PCR. The nucleotide sequence of the endonuclease gene and the methylase gene were determined. The NlaIII endonuclease gene is 693 bp, encoding a protein with predicted molecular weight of 26487. The NlaIII methylase gene was identical with that previously reported [Labbe, D., Joltke, H.J. and Lau, P.C. (1990) Cloning and characterization of two tandemly arranged DNA methyltransferse genes of Neisseria lactamica: an adenine-specific M.NlaIII and a cytosine-type methylase. Mol. Gen. Genet. 224, 101-110]. The endonuclease and methylase genes overlap by four bases and are transcribed in the same orientation. The endonuclease gene was cloned into an improved T7 vector, and a high level of NlaIII endonuclease expression was achieved in E. coli.     

A novel glycerol kinase from Flavobacterium meningosepticum: characterization, gene cloning and primary structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

Cyclobutane thymine dimers with a disrupted phosphodiester bond are refractory to T4 endonuclease V digestion but have increased sensitivity to UvrABC nuclease

UV irradiation induces the dimerization of synthetic single-stranded, 80-mer oligonucleotides with self-complementary, alternating purine-pyrimidine sequences, and terminal 5'- and 3'-thymines; this process can be reversed by photoreactivation. The UV-induced 160-mers are sensitive to digestion by the restriction enzyme SnaBI, but monomers are insensitive to digestion, indicating that UV irradiation stabilizes the formation of double-stranded DNA. These results suggest that UV irradiation of these 80-mer oligonucleotide substrates induces the formation of a novel cyclobutane thymine dimer which lacks an intradimer phosphodiester bond (CPD*). This CPD*, linking the terminal thymines of two separate 80-mer molecules, is formed in a double-stranded DNA region created by self-annealing and intermolecular hybridization of the two 80-mer strands. We have found that these UV-induced CPD* in 160-mers are sensitive to cleavage by the nucleotide excision enzyme complex UvrABC nuclease, but resistant to cleavage by the cyclobutane pyrimidine dimer-specific enzyme T4 endonuclease V. However, pretreatment of the 160-mers with ligase reverses their sensitivity to these two enzymes, significantly reducing their susceptibility to cleavage by UvrABC nuclease but dramatically increasing their susceptibility to cleavage by T4 endonuclease. The biological significance of these findings is discussed.     

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes

Exposure of an aqueous, aerated solution (pH 7) of a double-stranded DNA to 193 nm light, of sufficient energy to ionise DNA, leads to selective, non-random modification at guanine in the form of frank single-strand break (ssb) and base modifications, revealed by treatment with either Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), Escherichia coli endonuclease III (Nth) or hot piperidine treatment. There is a similar neighbouring base sequence dependence for Fpg- and Nth-sensitive damage as that previously reported for both hot alkali-labile damage and prompt ssb. Low yields of photoproducts, namely pyrimidine dimers, are also revealed using the enzyme T4 endonuclease V (T4 endo V). Although irradiation of DNA with 193 nm light causes photoionisation of all the nucleic acid bases, these results indicate that guanine is the predominant site for localisation of the oxidative damage. These findings are consistent with migration of the radical cation to 'target' damage at guanine sites.     

Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants

Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.     

The fission yeast UVDR DNA repair pathway is inducible

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.     

Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

3-Ketosteroid-delta1-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme

The gene encoding 3-ketosteroid-Delta1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Delta1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Delta1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.     

An oxidative damage-specific endonuclease from rat liver mitochondria

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.     

Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI)

The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.     

A new class of DNA photolyases present in various organisms including aplacental mammals

DNA photolyase specifically repairs UV light-induced cyclobutane-type pyrimidine dimers in DNA through a light-dependent reaction mechanism. We have obtained photolyase genes from Drosophila melanogaster (fruit fly), Oryzias latipes (killifish) and the marsupial Potorous tridactylis (rat kangaroo), the first photolyase gene cloned from a mammalian species. The deduced amino acid sequences of these higher eukaryote genes show only limited homology with microbial photolyase genes. Together with the previously cloned Carassius auratus (goldfish) gene they form a separate group of photolyase genes. A new classification for photolyases comprising two distantly related groups is proposed. For functional analysis P.tridactylis photolyase was expressed and purified as glutathione S-transferase fusion protein from Escherichia coli cells. The biologically active protein contained FAD as light-absorbing cofactor, a property in common with the microbial class photolyases. Furthermore, we found in the archaebacterium Methanobacterium thermoautotrophicum a gene similar to the higher eukaryote photolyase genes, but we could not obtain evidence for the presence of a homologous gene in the human genome. Our results suggest a divergence of photolyase genes in early evolution.     

On the roles of urocanic acid in photoimmunosuppression: attempted photorepair of urocanic acid-DNA cyclobutane adducts with DNA photolyase

It has been reported that UV-induced immunosuppression can be reversed by photoreactivation or exposure to T4 endonuclease V, two treatments that can repair cyclobutane pyrimidine dimers. These observations, together with the known role of urocanic acid (UA) in UV-induced immune suppression, prompted us to study the ability of DNA photolyase to repair UA-DNA cyclobutane photoadducts in single-stranded calf thymus DNA. We did not detect any release of UA, with a sensitivity implying that photolyase is at least 2900 times less active toward UA-DNA adducts than toward cis-syn thymine-thymine dimers. This indicates that any reversal of photoimmunosuppression by photoreactivation cannot significantly involve cleavage of UA-DNA cyclobutane adducts.