Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli

The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of phi X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions. phi X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease III is involved in the repair of thymine glycols in vivo. phi X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease III and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols. phi X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease III or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other. These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV.     

Processing of model single-strand breaks in phi X-174 RF transfecting DNA by Escherichia coli

The inactivation efficiency and repair of single-strand breaks was investigated using model strand breaks created by endonucleolytic incision of damaged DNA. Phi X-174 duplex transfecting DNA containing either thymine glycols, urea residues, or abasic (AP) sites was incubated with AP endonucleases that produce breaks on the 3' side, the 5' side, or both sides of the lesion. For each lesion, incubation with Escherichia coli endonuclease III results in a single-strand break containing a 3' alpha, beta-unsaturated aldehyde (4-hydroxy-2-pentenal), while treatment of AP- or urea-containing DNA with E. coli endonuclease IV results in a single-strand break containing a 5' deoxyribose or a 5' deoxyribosylurea moiety, respectively. Incubation of lesion-containing DNA with both enzymes results in a base gap. Ligatable nicks containing 3' hydroxyl and 5' phosphate moieties were produced by subjecting undamaged DNA to DNase I. When the biological activity of these DNAs was assessed in wild-type cells, ligatable nicks were not lethal, but each of the other strand breaks tested was lethal, having inactivation efficiencies between 0.12 and 0.14. These inactivation efficiencies are similar to those of the base lesions from which the strand breaks were derived. In keeping with the current model of base excision repair, when phi X duplex DNA containing strand breaks with a blocked 3' terminus was transfected into an E. coli double mutant lacking the major 5' cellular AP endonucleases, a greater than twofold decrease in survival was observed. Moreover, when this DNA was treated with a 5' AP endonuclease prior to transfection, the survival returned to that of wild type. As expected, when DNA containing strand breaks with a 5' blocked terminus or DNA containing base gaps was transfected into the double mutant lacking 5' AP endonucleases, the survival was the same as in wild-type cells. The decreased survival of transfecting DNA containing thymine glycols, urea, or AP sites observed in appropriate base excision repair-defective mutants was also obviated if the DNA was incubated with the homologous enzyme prior to transfection. Thus, in every case, with both base lesions and single-strand breaks, the lesion was repaired in the cell by the enzyme that recognizes it in vitro. Furthermore, the repair step in the cell could be eliminated if the appropriate enzyme was added in vitro prior to transfection.(ABSTRACT TRUNCATED AT 400 WORDS)     

UvrABC nuclease complex repairs thymine glycol, an oxidative DNA base damage

The UvrABC nuclease complex recognizes a wide spectrum of DNA lesions including pyrimidine dimers, bulky chemical adducts and O6-methylguanine. In this study we have demonstrated that the UvrABC complex is also able to incise PM2 DNA containing the oxidative DNA lesion, thymine glycol. However, DNA containing dihydrothymine, a lesion with a similar structure to thymine glycol, was not incised. The UvrABC complex was also able to incise DNA containing reduced apurinic sites or apurinic sites modified with O-alkyl hydroxylamines, but not DNA containing apurinic sites or urea residues. In vivo, in the absence of base-excision repair, nucleotide excision repair was operable on phi X-174 RF transfecting DNA containing thymine glycols. The level of the repair was found to be directly related to the level of the UvrABC complex. Thus, UvrABC-mediated nucleotide excision repair appears to play a role in the repair of thymine glycol, an oxidative DNA-base lesion that is produced by ionizing radiation or formed during oxidative respiration.     

Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.     

Immunochemical quantitation of thymine glycol in oxidized and X-irradiated DNA

The present study demonstrates the usefulness of immunochemical assays for quantitating modified bases in oxidized and X-irradiated DNA. Escherichia coli, phi X174 RF I, PM2, and M13 DNA containing thymine glycols introduced by OsO4 oxidation were used as antigens in a direct enzyme-linked immunosorbent assay (ELISA). The number of thymine glycols per DNA molecule was determined by reactivity with antithymine glycol antibody standardized either to the acetol fragment assay or to the number of Escherichia coli endonuclease III-sensitive sites. The number of thymine glycols was also determined in phi X174 RF I DNA X-irradiated in either phosphate or Tris buffer under air. Using a direct ELISA with phi X174 RF I DNA irradiated in a phosphate buffer solution, the anti-thymine glycol antibody detected damage at the level of 40 Gy. The immunochemical assay was sensitive, specific, quantitative, and independent of DNA structure.     

Absence of a role for DNA polymerase II in SOS-induced translesion bypass of phi X174.

In order to examine the possible role of Escherichia coli DNA polymerase II in SOS-induced translesion bypass, Weigle reactivation and mutation induction were measured with single-stranded phi X174 transfecting DNA containing individual lesions. No decrease in bypass of thymine glycol or cyclobutane pyrimidine dimers in the absence of DNA polymerase II was observed. Furthermore, DNA polymerase II did not affect bypass of abasic sites when either survival or mutagenesis was the endpoint. Lastly, repair of gapped DNA molecules, intermediates in methyl-directed mismatch repair, was also unaffected by the presence or absence of DNA polymerase II.     

An international collaborative study of ''genetic drift'' in Salmonella typhimurium strains used in the Ames test

The efficiency of Weigle reactivation of ultraviolet light-irradiated single and double-stranded phi X174 DNA by wild-type and excision repair-defective E. coli hosts was determined. After limited exposure to ultraviolet light, the efficiency of Weigle reactivation by an ultraviolet light-irradiated wild-type host was greater for double-stranded phi X174 DNA than for its single-stranded counterpart. However, the efficiency of inducible recovery of the double-stranded DNA molecule decreased as its exposure to ultraviolet light increased until it became constant at a value 1.5 times less than that for single-stranded form of phi X174 DNA. The efficiency of Weigle reactivation of the single-stranded DNA molecule by the same host, however, was independent of the dose to the DNA, as were the efficiencies of reactivation for both forms of phi X174 DNA by ultraviolet light-irradiated excision repair-deficient hosts. In excision repair-defective hosts the efficiency of Weigle reactivation of double-stranded phi X174 DNA was also 1.5 times less than that for the single-stranded molecule. These results suggest that the Weigle reactivation of double-stranded phi X174 DNA is mediated in part by an excision repair process, and that this component of Weigle reactivation eventually can be saturated by ultraviolet light-induced DNA damage leaving other repair processes, such as trans-damage synthesis, responsible for the remaining inducible reactivation.     

Inducible repair of thymine ring saturation damage in phi X174 DNA

The susceptibility to inducible SOS repair of 5,6-dihydroxy-dihydrothymine (t') damage in single-stranded phi X174 DNA has been measured. Following exposure to osmium tetroxide, which introduces t' damage in DNA under the conditions used, biological survival of the DNA infected into spheroplasts of Escherichia coli which had received prior exposure to ultraviolet light was higher than in unexposed spheroplasts. From our results, we conclude that approximately 63% of the biological damage from t' products, which is one of the classes of damage present in DNA following ionizing radiation, is susceptible to repair by the inducible SOS repair system.     

Characterization of a wide range base-damage-endonuclease activity of mammalian rpS3

Mammalian rpS3, a ribosomal protein S3 with a DNA repair endonuclease activity, nicks heavily UV-irradiated DNA and DNA containing AP sites. RpS3 calls for a novel endonucleolytic activity on AP sites generated from pyrimidine dimers by T4 pyrimidine dimer glycosylase activity. This study revealed that rpS3 cleaves the lesions including AP sites, thymine glycols, and other UV damaged lesions such as pyrimidine dimers. This enzyme does not have a glycosylase activity as predicted from its amino acid sequence. However, it has an endonuclease activity on DNA containing thymine glycol, which is exactly overlapped with UV-irradiated or AP DNAs, indicating that rpS3 cleaves phosphodiester bonds of DNAs containing altered bases with broad specificity acting as a base-damage-endonuclease. RpS3 cleaves supercoiled UV damaged DNA more efficiently than the relaxed counterpart, and the endonuclease activity of rpS3 was inhibited by MgCl2 on AP DNA but not on UV-irradiated DNA.     

Mechanism of action of Escherichia coli endonuclease III

Endonuclease III isolated from Escherichia coli has been shown to have both N-glycosylase and apurinic/apyrimidinic (AP) endonuclease activities. A nicking assay was used to show that the enzyme exhibited a preference for form I DNA when DNA containing thymine glycol was used as a substrate. This preference was reduced or eliminated either when the DNA was relaxed or when the type of damage was altered to urea residues or AP sites. The combined N-glycosylase/AP endonuclease activity was at least 10-fold higher than the AP endonuclease activity alone when urea-containing DNA was used as a substrate as compared to AP DNA. When DNA containing thymine glycol was used as a substrate, the combined N-glycosylase/AP endonuclease activity was about 2-fold higher than the AP endonuclease activity. Yet, when DNA containing thymine glycol or urea was used as substrate, no apurinic sites remained. Furthermore, magnesium selectively inhibited endonuclease III activity when AP DNA was used as a substrate but had no effect when DNA containing either urea or thymine glycol was used as substrate. These data suggest that both the N-glycosylase and AP endonuclease activities of endonuclease III reside on the same molecule or are in very tight association and that these activities act in concert, with the N-glycosylase reaction preceding the AP endonuclease reaction.     

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.     

Melanin both causes and prevents oxidative base damage in DNA: quantification by anti-thymine glycol antibody.

The present study employs immunological methods to measure modified bases in DNA. A polyclonal antibody specific for thymine glycol was used to quantify the level of thymine glycol in calf thymus DNA gamma-irradiated in solutions containing varying concentrations of DOPA-eumelanin. Melanin decreased the number of thymine glycols produced by 200 Gy at low melanin concentrations. At high melanin concentrations, the number of thymine glycols increased. Thymine glycol was also produced in unirradiated DNA-eumelanin mixtures. DOPA-eumelanin was found to produce single-strand breaks in supercoiled phi X174 RF DNA. The breaks were measured by conversion of form I to form II as detected by agarose gel electrophoresis. The level of damage produced by melanin could be modulated by agents known either to stabilize or to scavenge active oxygen species. These studies demonstrate that melanin can both scavenge and generate active free radicals.     

Mechanism of action of Micrococcus luteus gamma-endonuclease

Micrococcus luteus extracts contain gamma-endonuclease, a Mg2+-independent endonuclease that cleaves gamma-irradiated DNA. This enzyme has been purified approximately 1000-fold, and the purified enzyme was used to study its substrate specificity and mechanism of action. gamma-Endonuclease cleaves DNA containing either thymine glycols, urea residues, or apurinic sites but not undamaged DNA or DNA containing reduced apurinic sites. The enzyme has both N-glycosylase activity that releases thymine glycol residues from OsO4-treated DNA and an associated apurinic endonuclease activity. The location and nature of the cleavage site produced has been determined with DNA sequencing techniques. gamma-Endonuclease cleaves DNA containing thymine glycols or apurinic sites immediately 3' to the damaged or missing base. Cleavage results in a 5'-phosphate terminus and a 3' baseless sugar residue. Cleavage sites can be converted to primers for DNA polymerase I by subsequent treatment with Escherichia coli exonuclease III. The mechanism of action of gamma-endonuclease and its substrate specificity are very similar to those identified for E. coli endonuclease III.     

Analysis of the activity of Micrococcus luteus endonucleases with respect to gamma-irradiated DNA]

Endonucleases from Micrococcus luteus that induce single-strand breaks in gamma-irradiated DNA have been separated chromatographycally into two groups. The first group involves two different enzymes: AP-endonuclease II (mol. weight 30 000) and AP, UV-endonuclease I (mol. weight 15 000) that recognize alkali-labile lesions in gamma-irradiated DNA and apurinic sites in DNA heated at 70 degrees C, pH 6.08 AP-endonuclease II in cooperation with DNA polymerase from M. luteus and T4 phage-induced polynucleotide ligase is capable of carrying out in vitro complete excision repair of alkali-labile lesins in gamma-irradiated DNA. The second group involves gamma-endonucleases X and Y that act on alkalistable gamma-ray lesions. gamma-endonucleases X and Y can be separated by chromatography on DEAE-cellulose but possess similar properties. Activity of gamma-endonucleases toward gamma-irradiated DNA is inhibited by only heavily UV-irradiated DNA (15 000 ergs/mm2). The data are consistent with the hypothesis that gamma-endonucleases are specific for thymine glycols (t' and tUV) in UV- and gamma-irradiated DNA.     

Excision repair participates in the Weigle reactivation of ultraviolet light-irradiated φ X174 double-stranded DNA

The efficiency of Weigle reactivation of ultraviolet light-irradiated single and double-stranded φX174 DNA by wild-type and excision repair-defective E. coli hosts was determined. After limited exposure to ultraviolet light, the efficiency of Weigle reactivation by an ultraviolet light-irradiated wild-type host was greater for double-stranded φX174 DNA than for its single-stranded counterpart. However, the efficiency of inducible until it became constant at a value 1.5 times less than that for single-stranded form of φX174 DNA. The efficiency of Weigle reactivation of the single-stranded DNA molecule by the same host, however, was independent of the dose to the DNA, as were the efficiencies of reactivation for both forms of φX174 DNA by ultraviolet light-irradiated excision repair-deficient hosts. In excision repair-defective hosts the efficiency of Weigle reactivation of double-stranded φX174 DNA was also 1.5 time less than that for the single-stranded molecule.These results suggest that the Weigle reactivation of double-stranded φX174 DNA is mediated in part by an excision repair process, and that this component of Weigle reactivation eventually can be saturated by ultraviolet light-induced DNA damage leaving other repair processes, such as trans-damage synthesis, responsible for the remaining inducible reactivation.     

Compromised incision of oxidized pyrimidines in liver mitochondria of mice deficient in NTH1 and OGG1 glycosylases

Mitochondrial DNA is constantly exposed to high levels of endogenously produced reactive oxygen species, resulting in elevated levels of oxidative damaged DNA bases. A large spectrum of DNA base alterations can be detected after oxidative stress, and many of these are highly mutagenic. Thus, an efficient repair of these is necessary for survival. Some of the DNA repair pathways involved have been characterized, but others are not yet determined. A DNA repair activity for thymine glycol and other oxidized pyrimidines has been described in mammalian mitochondria, but the nature of the glycosylases involved in this pathway remains unclear. The generation of mouse strains lacking murine thymine glycol-DNA glycosylase (mNTH1) and/or murine 8-oxoguanine-DNA glycosylase (mOGG1), the two major DNA N-glycosylase/apurinic/apyrimidinic (AP) lyases involved in the repair of oxidative base damage in the nucleus, has provided very useful biological model systems for the study of the function of these and other glycosylases in mitochondrial DNA repair. In this study, mouse liver mitochondrial extracts were generated from mNTH1-, mOGG1-, and [mNTH1, mOGG1]-deficient mice to ascertain the role of each of these glycosylases in the repair of oxidized pyrimidine base damage. We also characterized for the first time the incision of various modified bases in mitochondrial extracts from a double-knock-out [mNTH1, mOGG1]-deficient mouse. We show that mNTH1 is responsible for the repair of thymine glycols in mitochondrial DNA, whereas other glycosylase/AP lyases also participate in removing other oxidized pyrimidines, such as 5-hydroxycytosine and 5-hydroxyuracil. We did not detect a backup glycosylase or glycosylase/AP lyase activity for thymine glycol in the mitochondrial mouse extracts.     

Repair of dihydrouracil supported by base excision repair in mNTH1 knock-out cell extracts

In mammalian cells, thymine glycols and other oxidized pyrimidines such as 5,6-dihydrouracil are removed from DNA by the NTH1 protein, a bifunctional DNA-N-glycosylase. However, mNTH1 knock-out mice in common with other DNA glycosylase-deficient mice do not show any severe abnormalities associated with accumulation of DNA damage and mutations. In the present study we used an in vitro repair system to investigate the mechanism for the removal of 5,6-dihydrouracil from DNA by mNTH1-deficient cell-free extracts derived from testes of mNTH1 knock-out mice. We found that these extracts are able to support the removal of 5,6-dihydrouracil from DNA at about 20% of the efficiency of normal extracts. Furthermore, we also found that single-nucleotide patch base excision repair is the major pathway for removal of 5,6-dihydrouracil in mNTH1-deficient cell extracts, suggesting the involvement of other DNA glycosylase(s) in the removal of oxidized pyrimidines.     

Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic

Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. The potential for genetic change arising from the effects of clustered damage sites containing combinations of AP sites, 8-oxo-7,8-dihydroguanine (8-oxoG) or 5,6-dihydrothymine is high. To date clusters containing a DNA base lesion that is a strong block to replicative polymerases, have not been explored. Since thymine glycol (Tg) is non-mutagenic but a strong block to replicative polymerases, we have investigated whether clusters containing Tg are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxoG or an AP site. Using a bacterial plasmid-based assay and repair assays using cell extracts or purified proteins, we have shown that DNA double-strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted base excision repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster ‘protects’ against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG, due to the decisive role of endonucleases in the initial stages of processing Tg/8-oxoG clusters, removing Tg to give an intermediate with an abasic site or single-strand break.     

Effects of SOS and MucAB functions on reactivation and mutagenesis of M13 replicative form DNA bearing bulky lesions

We have previously determined the specificity of -1 frameshifts induced by aflatoxin-B1-2,3-dichloride (AFB1C12) in phage M13 double-strand replicative form (RF) DNA. The system consists of: (i) in vitro adduction of RF DNA of BK8, a lacZ + 1 frameshift derivative of phage M13mp8; (ii) transfection into unirradiated or UV-irradiated bacterial host cells; (iii) scoring and sequencing of revertants (i.e., -1 frameshifts). The previous data had shown that induction of SOS functions enhanced mutagenesis. However, this increase in mutagenesis is not accompanied by enhanced survival in a majority of the strains tested. Here, we present evidence to show that the lack of SOS reactivation is a specific property of the RF DNA system rather than a specific property of the lesion. A model mechanism based on the replicative strategy of transfected RF DNA can account for these observations. In addition, we have calculated individual Weigle mutagenesis factors at 8 major mutagen induced sites reported previously. Analysis of these data indicates that, within a restricted subset of possible mutational events (i.e., -1 frameshifts), Weigle mutagenesis is affected by both the DNA sequence environment of the mutation site as well as the repair phenotype of the cell.