A novel role for Escherichia coli endonuclease VIII in prevention of spontaneous G-->T transversions.

In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site. To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.

In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme.

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.

In search of a mutational hotspot.

In vitro selection was used to define sequence contexts that significantly enhanced the mutagenic potential of 7, 8-dihydro-8-oxoguanine (8-oxoG). Contexts that simultaneously reduced the efficiency of 8-oxoG cleavage by formamidopyrimidine DNA N-glycosylase and increased the efficiency of misincorporating A opposite the lesion by DNA polymerase were isolated from a pool of 4(8) random octanucleotide sequences. Kinetic analysis showed that the combined effects of poor repair and high miscoding resulted in 10(2)- to 10(3)-fold increase in the mutagenic potential of 8-oxoG. Furthermore, the isolated sequence contexts correlated strongly with G --> T transversion hotspots in spontaneous mutational spectra reported for the Escherichia coli lacI and human p53 and factor IX genes. We present an example directly linking the interplay between DNA repair and replication to a "high risk sequence" for base substitution.

Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine.

A major stable oxidation product of DNA cytosine is uracil glycol (Ug). Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties. Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo-). During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined. Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg. Ug opposite G and A was also recognized and removed in vitro by the E. coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase. The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate.

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII.

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.

Asbestos increases mammalian AP-endonuclease gene expression, protein levels, and enzyme activity in mesothelial cells.

Only two DNA repair enzymes, DNA polymerase beta and O6-methylguanine-DNA methyltransferase, have been shown to be inducible in mammalian cells by genotoxic agents. We show here that crocidolite asbestos induces the DNA repair enzyme, apurinic/apyrimidinic (AP)-endonuclease, in isolated mesothelial cells, the progenitor cells of malignant mesothelioma. Asbestos at nontoxic concentrations of 1.25 and 2.5 microg/cm2 significantly increased AP-endonuclease mRNA and protein levels as well as enzyme activity (P < 0.05) in a dose-dependent manner in rat pleural mesothelial cells. These increases were persistent from 24 to 72 h after initial exposure to fibers. Changes were not observed with glass beads, a noncarcinogenic particle. Confocal scanning laser microscopy showed that AP-endonuclease was primarily localized in the nucleus but also in mitochondria. Our data are the first to demonstrate the inducibility of AP-endonuclease by a human class I carcinogen associated with oxidant stress in normal cells of the lung.

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.

Characterization of Escherichia coli endonuclease VIII.

Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (AP) sites (Melamede, R. J., Hatahet, Z., Kow, Y. W., Ide., H., and Wallace, S. S. (1994) Biochemistry 33, 1255-1264). Using apparently homogeneous endo VIII protein, we now show that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil. Endo VIII cleaved the damage-containing DNA strand by beta,delta-elimination as does formamidopyrimidine DNA glycosylase (Fpg). Like Fpg, endo VIII also excised the 5'-terminal deoxyribose phosphate from an endonuclease IV (endo IV) pre-incised AP site. Thus, in addition to amino acid sequence homology (Jiang, D., Hatahet, Z., Blaisdell, J., Melamede, R. J., and Wallace, S. S. (1997) J. Bacteriol. 179, 3773-3782), endo VIII shares a number of catalytic properties with Fpg. In addition, endo VIII specifically bound to oligodeoxynucleotides containing a reduced AP site with a stoichiometry of 1:1 for protein to DNA with an apparent equilibrium dissociation constant of 3.9 nM. Like Fpg and endo III, the DNase I footprint was small with contact sites primarily on the damage-containing strand; unlike Fpg and endo III, the DNA binding of endo VIII to DNA was asymmetric, 3' to the reduced AP site.

Validation of a simple method assessing nitric oxide and nitrogen dioxide concentrations.

Monitoring of nitric oxide (NO) and nitrogen dioxide (NO2) is a prerequisite for the clinical use of NO. Chemiluminescence, the reference method, cannot be used as a routine in clinical practice in view of its cost and other restraints. This study was performed to evaluate a device using an electrochemical method (Polytrons NO and NO2, Dräger). Forty-nine simultaneous measurements of NO and various oxides of nitrogen (NOx) concentrations by the two apparatus were performed. NO measurements by means of these two methods are very well correlated (r = 0.96; p < 10(-5)). The mean difference according to the method of Bland and Altman was 2.8 +/- 1.7 ppm, with the limits of agreement at -0.6 and +6.2 ppm (confidence interval of 95%). There was also a good correlation between measurements of NO2 obtained via Polytrons and NOx via chemiluminescence (r = 0.84; p < 10(-5)). However, NO2 measurements obtained via Polytrons may be insufficient to exclude potential toxicity of NO2 due to the inability to detect measurements in the ppb-range. This study demonstrates that devices designed for industrial purposes (Polytrons NO and NO2, Dräger) can be used for clinical purposes.

Single lung transplantation for pulmonary lymphangiomyomatosis. Unexpected need for extracorporeal circulation.

The present case describes an acute respiratory-related hemodynamic failure during a single left lung transplantation in a 32-year-old woman suffering from end-stage pulmonary lymphangiomyomatosis. During the first 5 min of single right lung ventilation, a progressive increase in airway pressure and decrease in tidal volume associated with a decrease in arterial pressure and Spo2 occurred that were successfully countered by reventilation of the left lung. Proper positioning of the double-lumen tube was confirmed with a fiberoptic bronchoscope. Despite deliberate hypoventilation, within a few respiratory cycles, each further attempt at single lung ventilation was followed by abrupt hypotension, increase in pulmonary artery pressure, while airway pressure rose and tidal volume collapsed. The surgical team saw no signs of right pneumothorax. In these circumstances, cardiopulmonary bypass was required to perform pneumonectomy and grafting. Postoperatively a right anterior pneumothorax remained undiscovered on standard radiograph but was later revealed on soft radiograph. This acute intraoperative respiratory failure could equally well have been related to air trapping, in which case, however, deliberate hypoventilation would have been effective. In addition, the striking difference between the progressive onset of the first episode of hemodynamic failure and the immediate onset of the others argues in favor of a pneumothorax being at cause. Patients with pulmonary lymphangiomyomatosis are at high risk for intraoperative pneumothorax, but in our case, it could not be confirmed and treated during the surgical procedure without putting the patient at high risk for lung injury because of pleurodesis due to earlier pleural abrasion. This case again clearly shows the need to have cardiopulmonary bypass whenever single lung transplantation is performed.

New substrates for old enzymes. 5-Hydroxy-2'-deoxycytidine and 5-hydroxy-2'-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2'-deoxyuridine is a substrate for uracil DNA N-glycosylase.

5-Hydroxy-2'-deoxycytidine (5-OHdC) and 5-hydroxy-2'-deoxyuridine (5-OHdU) are major products of oxidative DNA damage with mutagenic potential. Until now, no enzymatic activity responsible for their removal has been identified. We report here that both 5-OHdC and 5-OHdU are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase (FPG). 5-OHdU is also a substrate for uracil DNA N-glycosylase. Consistent with their mechanisms of action on previously described substrates, endonuclease III removes 5-OHdC and 5-OHdU via a N-glycosylase/beta-elimination reaction, FPG follows a N-glycosylase/beta,delta-elimination reaction, and uracil N-glycosylase removes 5-OHdU by N-glycosylase action leaving behind an abasic site. Endonuclease III removes both lesions more efficiently than FPG, and both endonuclease III and FPG remove 5-OHdC slightly more efficiently than 5-OHdU. Uracil DNA N-glycosylase removes 5-OHdU more efficiently than the other two enzymes and has no activity on 5-OHdC even when present in great excess. Analysis of crude extracts obtained from wild type and endonuclease III deletion mutants of E. coli correlated well with data obtained with the purified enzymes.

Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli.

We have purified a novel endonuclease from Escherichia coli that recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA. This activity, which we named deoxyinosine 3' endonuclease, is different from the known hypoxanthine DNA N-glycosylases which have been partially characterized in E. coli and other organisms. The enzyme was purified 24,800-fold to apparent homogeneity. SDS- and activity PAGE analyses indicate that the enzyme has an apparent molecular mass of 25 kDa. Deoxyinosine 3' endonuclease recognized deoxyinosine in both single- and double-stranded DNA but exhibited a 4-fold preference for double stranded over single-stranded DNA. In addition to deoxyinosine, the enzyme recognized urea residues and AP sites. Deoxyinosine 3' endonuclease has an obligatory requirement for Mg2+, but other cations such as Co2+ and Mn2+ could partially replace Mg2+. The optimal pH for deoxyinosine 3' endonuclease was around 7.5. In contrast to most of the known repair enzymes, deoxyinosine 3' endonuclease makes an incision at the second phosphodiester bond 3' to a deoxyinosine or AP site, leaving behind the intact lesion on the nicked DNA. Therefore, deoxyinosine 3' endonuclease recognizes, but does not remove, the lesion from the DNA molecule. The biological significance of this novel activity is discussed with reference to other repair activities in E. coli.

Isolation and characterization of endonuclease VIII from Escherichia coli.

Endonuclease VIII, a novel presumptive DNA repair enzyme, was isolated from Escherichia coli by FPLC1 purification. The enzyme was found in strains that contained or lacked endonuclease III and was purified by radial flow S-Sepharose, Mono S, phenyl-Superose, and Superose 12 FPLC. Examination of the properties of endonuclease VIII showed it to have many similarities to endonuclease III. DNA containing thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, urea residues, or AP sites was incised by the enzyme; however, DNA containing reduced AP sites was not. HPLC analysis of the products formed by exhaustive enzymatic digestion of damage-containing DNA showed that endonuclease VIII released thymine glycol and dihydrothymine as free bases. Taken together, these data suggest that endonuclease VIII contains both N-glycosylase and AP lyase activities. Consistent with this idea, DNA containing AP sites or thymine glycols, that was enzymatically nicked by endonuclease VIII was not a good substrate for E. coli DNA polymerase I, suggesting that endonuclease VIII nicks damage-containing DNA on the 3' side of the lesion. Also, since monophosphates were not released after treating thymine glycol-containing DNA with endonuclease VIII, the enzyme does not appear to have exonuclease activity. The enzyme activity was maximal in 75 mM NaCl or 5 mM MgCl2. Analysis of endonuclease VIII by both Superose FPLC and Sephadex yielded native molecular masses of 28,000 and 30,000 Da, respectively. SDS-PAGE, in conjunction with activity gel analysis, gave a molecular mass of about 29,000 Da. Furthermore, renaturation of the putative active band from SDS-PAGE gave rise to an active enzyme.

Pyrimidine ring fragmentation products. Effects of lesion structure and sequence context on mutagenesis.

Free radicals produce a broad spectrum of damages to DNA, a major proportion of which includes ring fragmentation and contraction products of DNA bases as well as abasic sites. In this study, the mutagenic potential fo two pyrimidine ring fragmentation products, urea and beta-ureidoisobutyric acid (UBA), was analyzed using the i-d region of the Escherichia coli lacI gene contained in a single-stranded f1-K12 phage hybrid vector. Single-stranded DNA was used so that the in vivo interactions between the damage and the DNA polymerase could be assessed in the absence of excision repair. The i-d region contains 20 mutable thymine sites so that 20 separate sequence contexts containing a unique lesion at a position of thymine can be analyzed simultaneously. Urea and UBA residues were uniquely introduced into f1-K12 DNA by chemical and enzymatic methods and primer extension and piperidine analysis of the damage-containing template molecules demonstrated that the potential mutable thymine sites contained randomly distributed lesions. Both fragmentation products were poorly bypassed by DNA polymerases in vitro and in the cell; although in the presence of SOS-induction, UBA was bypassed more efficiently than urea. UBA was a potent premutagenic lesion with a rate of mutation induction more than sixfold above that observed with abasic sites derived from purines. Urea residues were about as mutagenic as abasic sites derived from purines, which in turn were more mutagenic than abasic sites derived from thymine. Mutations derived from urea, UBA and abasic sites were all dependent on SOS-induction of the host cells. Since both urea and UBA were derived from DNA thymine, these data demonstrate that adenine is not routinely inserted opposite products that no longer retain the structural integrity of the pyrimidine ring. Sequence analysis showed that the mutations were targeted at thymine with 62% of the urea-derived mutations being T to C transitions and 62% of the UBA derived mutations being T to A transversions. Thus, the two fragmentation products appeared to direct specific misinsertions. The mutations were not randomly distributed over the i-d region for either fragmentation product and hotspots were observed for both damages. The presence of hotspots suggests that in addition to lesion structure, sequence context plays an important role in base selectivity by DNA polymerases opposite DNA lesions. Energy minimization calculations were used to model the urea and UBA lesions at two contrasting hotspot sites. In both cases, there was significant agreement between the computational and biological data sets.

Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis.

We have used thymine glycol and dihydrothymine as representative ring saturation products resulting from free-radical interaction with DNA pyrimidines, and urea glycosides and beta-ureidoisobutyric acid (UBA) as models for pyrimidine-ring fragmentation products. We have shown that thymine glycol and the ring-fragmentation products urea and beta-ureidoisobutyric acid, as well as abasic sites, are strong blocks to DNA polymerases in vitro. In contrast, dihydrothymine is not a block to any of the polymerases tested. For thymine glycol, termination sites were observed opposite the putative lesions, whereas for the ring-fragmentation products, the termination sites were primarily one base prior to the lesion. These and other data have suggested that thymine glycol codes for an A, and that a base is stably inserted opposite the damage, whereas when a base is inserted opposite the non-coding lesions, it is removed by the 3-->5 exonuclease activity of DNA polymerase I. Despite their efficiency as blocking lesions, thymine glycol, urea and UBA can be bypassed at low frequency in certain specific sequence contexts. When the model lesions were introduced individually into single-stranded biologically active DNA, we found that thymine glycol, urea, beta-ureidoisobutyric acid, and abasic sites were all lethal lesions having an activation efficiency of 1, whereas dihydrothymine was not. Thus the in vitro studies predicted the in vivo results. When the survival of biologically active single-stranded DNA was examined in UV-induced Escherichia coli cells where the block to replication was released, no increase in survival was observed for DNA containing urea or abasic sites, suggesting inefficient bypass of these lesions. In contrast, beta-ureidoisobutyric acid survival was slightly enhanced, and transfecting DNA containing thymine glycols was significantly reactivated. When mutation induction by unique lesions was measured using f1-K12 hybrid DNA containing an E. coli target gene, thymine glycols and dihydrothymine were found to be inefficient as premutagenic lesions, suggesting that in vivo, as in vitro, they primarily code for A. In contrast, urea and beta-ureidoisobutyric acid were efficient premutagenic lesions, with beta-ureidoisobutyric acid being about 4-5-fold more effective than urea glycosides, which have approximately the same rate of mutation induction as abasic sites from purines. Sequence analysis of the mutations resulting from these ring-fragmentation products shows that the mutations produced are both lesion and sequence context dependent. The possible roles that bypass efficiency and lesion-directed misinsertion might play in mutagenesis are discussed.

A novel method for site specific introduction of single model oxidative DNA lesions into oligodeoxyribonucleotides.

Calf thymus terminal deoxynucleotidyl transferase was used to incorporate several products of oxidative base damage onto the 3' end of oligodeoxyribonucleotides. Under the defined conditions described in this report, single residues of dihydrothymine, beta-ureidoisobutyric acid, thymine glycol, urea, 7-hydro-8-oxoadenine, 7-hydro-8-oxoguanine, 5-hydroxycytosine and 5-hydroxyuracil were incorporated into oligodeoxyribonucleotides of different lengths. The reaction is both efficient and cost effective. The 3' termini of the reaction products were suitable substrates for ligation by phage T4 DNA ligase, facilitating greatly the construction of oligodeoxyribonucleotides containing unique and site specific oxidative DNA lesions.