Cloning and characterization of an ascidian homolog of the human 8-oxoguanine DNA glycosylase (Ogg1) that is involved in the repair of 8-oxo-7,8-dihydroguanine in DNA in Ciona intestinalis.

PURPOSE: It is of interest to perform a systematic comparative analysis of the conserved domains in DNA glycosylases and the evolution of DNA base excision repair systems. Furthermore, it is important to characterize the roles and regulation of base excision repair during the development of organisms. To address these issues, we first identified 8-oxo-7,8-dihydroguanine (8-oxoG)-DNA glycosylase (Ogg1) of the ascidian Ciona intestinalis as a good model system. MATERIALS AND METHODS: A cDNA clone coding for a peptide with homology to human Ogg1 was identified in the expressed sequence tag (EST) database from the Ciona cDNA resources. We examined whether CiOgg1 has DNA glycosylase/AP (apurinic/apyrimidinic) lyase activities for 8-oxoG-containing oligonucleotide. Furthermore, the expression level of CiOgg1 was compared in various tissues of Ciona intestinalis. RESULTS: The CiOgg1gene encoded a protein of 351 amino acids, which shows 37% identity of amino acid sequence with human Ogg1. The Helix-hairpin-Helix motif was highly conserved. The ascidian enzyme had functional 8-oxoG-DNA glycosylase/AP lyase activities, which removed 8-oxoG opposite cytosine from DNA. Expression of the CiOgg1 significantly reduced the frequency of spontaneous G:C to T:A transversions in E. coli mutM mutY. The highest expression level was observed in testis in Ciona intestinalis. CONCLUSIONS: The structure and functions of Ogg1 are well conserved in Ciona intestinalis. CiOgg1 is involved in the repair of 8-oxoG in DNA in Ciona intestinalis.

Increased sensitivity to sparsely ionizing radiation due to excessive base excision in clustered DNA damage sites in Escherichia coli.

PURPOSE: In order to clarify the cellular processing and repair mechanisms for radiation-induced clustered DNA damage, we examined the correlation between the levels of DNA glycosylases and the sensitivity to ionizing radiation in Escherichia coli. MATERIALS AND METHODS: The lethal effects of gamma-rays, X-rays, alpha-particles and H2O2 were determined in E. coli with different levels of DNA glycosylases. The formation of double-strand breaks by post-irradiation treatment with DNA glycosylase was assayed with gamma-irradiated plasmid DNA in vitro. RESULTS: An E. coli mutM nth nei triple mutant was less sensitive to the lethal effect of sparsely ionizing radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of MutM (8-oxoguanine-DNA glycosylase), Nth (endonuclease III) and Nei (endonulease VIII) increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Increased sensitivity to gamma-rays also occurred in E. coli overproducing human 8-oxoguanine-DNA glycosylase (hOgg1). Treatment of gamma-irradiated plasmid DNA with purified MutM converted the covalently closed circular to the linear form of the DNA. On the other hand, overproduction of MutM conferred resistance to H2O2 on the E. coli mutM nth nei mutant. CONCLUSIONS: The levels of DNA glycosylases affect the sensitivity of E. coli to gamma-rays and X-rays. Excessive excision by DNA glycosylases converts nearly opposite base damage in clustered DNA damage to double-strand breaks, which are potentially lethal.

Ntg1 and Ntg2 proteins as 5-formyluracil-DNA glycosylases/AP lyases in Saccharomyces cerevisiae.

PURPOSE: 5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The present authors reported previously that MutM, Nth and Nei in Escherichia coli removed 5-foU from DNA. The present study identified 5-foU DNA glycosylases in Saccharomyces cerevisiae in order to clarify the repair mechanisms of 5-foU in eukaryotic cells. MATERIALS AND METHODS: The borohydride-trapping assay and DNA-nicking assay were carried out to detect and characterize the repair activities for 5-foU in extracts from S. cerevisiae with oligonucleotides containing 5-foU at specific sites. RESULTS: Two proteins in crude extracts from S. cerevisiae formed covalent complexes with oligonucleotides containing site-specific 5-foU in the presence of NaBH4. Extracts from S. cerevisiae strains defective in either the NTG1 or the NTG2 gene lacked either one or the other of these two proteins. Purified Ntg1 and Ntg2 were trapped in such complexes by the 5-foU-containing oligonucleotides in the presence of NaBH4. Furthermore, purified Ntg1 and Ntg2 efficiently cleaved the oligonucleotide at the 5-foU site. CONCLUSIONS: The results indicate that both Ntg1 and Ntg2 are involved in the repair of 5-foU in DNA, and thereby serve to reduce mutations in S. cerevisiae.

Strong static magnetic field and the induction of mutations through elevated production of reactive oxygen species in Escherichia coli soxR.

PURPOSE: Although strong static magnetic fields (SMF) are supposed to have the potential to affect biological systems, the effects have not been evaluated sufficiently. Experiments should be performed with a powerful SMF-generating apparatus to evaluate the biological effects of SMF. MATERIALS AND METHODS: An Escherichia coli mutation assay was used to assess the mutagenic effects of strong SMF. Various mutant strains of E. coli were exposed to up to 9 Tesla (T) for 24 h and the frequencies of rifampicin-resistant mutations were then determined. The expression of the soxS::lacZ fusion gene was assessed by measurement of beta-galactosidase activity. RESULTS: The results for survival or mutation were obtained with wild-type E. coli strain GC4468 and its derivatives defective in DNA repair enzymes or redox-regulating enzymes were all negative. On the other hand, the mutation frequency was significantly increased by the SMF exposure in soxR and sodAsodB mutants, which are defective in defence mechanisms against oxidative stress. Furthermore, the expression of superoxide-inducible soxS::lacZ fusion gene was stimulated 1.4- and 1.8-fold in E. coli when exposed to 5 and 9 T, respectively. CONCLUSIONS: These results indicate that strong SMF induce mutations through elevated production of intracellular superoxide radicals in E. coli.

Characterization of 2-hydroxyadenine DNA glycosylase activity of Escherichia coli MutY protein.

PURPOSE: 2-Hydroxyadenine (2-ohA) is an oxidation product of adenine generated in DNA by ionizing radiation and various chemical oxidants. 2-ohA has mutational potential comparable to that of 8-oxoguanine in bacteria and mammalian cells. Recent studies have shown that 2-ohA is removed from DNA by a human MutY homolog, MYH protein, in vitro. On the other hand, the repair mechanisms for 2-ohA in Escherichia coli are not yet understood. MATERIALS AND METHODS: Gel shift assays were used to assess the binding activity of E. coli full-length MutY protein and its N-terminal (residues 1-226) domain (M25) to 2-ohA/G-, 2-ohA/A-, 2-ohA/C- and 2-ohA/T-containing 24-mer oligonucleotides. Furthermore, whether these proteins specifically cleave 2-ohA-containing duplex oligonucleotides was examined. RESULTS: The purified MutY and M25 proteins had similar binding affinities to 2-ohA/G-, 2-ohA/A- and 2-ohA/C-containing oligonucleotides. MutY protein removed 2-ohA preferentially from 2-ohA/G mispairs. M25 protein showed the reduced catalytic activity for 2-ohA/G-containing oligonucleotides. CONCLUSIONS: E. coli MutY protein has a DNA glycosylase activity that removes 2-ohA from 2-ohA/G mispairs in DNA. The C-terminal domain is required for the removal of 2-ohA from DNA, but is not crucial for binding to 2-ohA-containing oligonucleotides.

Escherichia coli Nth and human hNTH1 DNA glycosylases are involved in removal of 8-oxoguanine from 8-oxoguanine/guanine mispairs in DNA.

The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C-->C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.

Mutagenic effects of 5-formyluracil on a plasmid vector during replication in Escherichia coli.

PURPOSE: 5-Formyluracil (5-foU) is a major derivative of thymine produced in DNA by ionizing radiation and various chemical oxidants. It has been previously shown that 5-foU in template DNA directs misincorporation of nucleotides by DNA polymerases during in vitro DNA synthesis. The present experiments were designed to understand the biological effects of5-foU in vivo. MATERIALS AND METHODS: The modified base was incorporated site-specifically into the recognition site of restriction endonuclease SalI (5'-GTCGAC) or AflII (5'-CTTAAG) in vector plasmid pSVK3 and introduced the plasmid into Escherichia coli. RESULTS: When the plasmids were replicated in E. coli, 5-foU caused mutations at the target sites. The induced mutation frequencies were 0.038-0.049%. Sequence analysis revealed that 5-foU preferentially caused T:A-->C:G and T:A-->A:T base substitutions and -1 deletions at the 5-foU site. 5-FoU also caused mutations at sites near the 5-foU. The alkA mutation did not affect the frequency of mutations in 5-foU-containing plasmids. CONCLUSIONS: The present experiments demonstrated that 5-formyluracil in DNA caused mutations in E. coli.

Identification of repair enzymes for 5-formyluracil in DNA. Nth, Nei, and MutM proteins of Escherichia coli.

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood. In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites. The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides. Extracts from strains defective in the nth, nei, or mutM gene lacked one of the proteins. All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant. The introduction of a plasmid carrying the nth, nei, or mutM gene into the E. coli triple mutant restored the formation of the corresponding protein-DNA complex. Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH(4). Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site. In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E. coli. The mutation frequency of the plasmid was significantly increased in the E. coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains. From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli.

Identification of proteins of Escherichia coli and Saccharomyces cerevisiae that specifically bind to C/C mismatches in DNA.

The pathways leading to G:C-->C:G transversions and their repair mechanisms remain uncertain. C/C and G/G mismatches arising during DNA replication are a potential source of G:C-->C:G transversions. The Escherichia coli mutHLS mismatch repair pathway efficiently corrects G/G mismatches, whereas C/C mismatches are a poor substrate. Escherichia coli must have a more specific repair pathway to correct C/C mismatches. In this study, we performed gel-shift assays to identify C/C mismatch-binding proteins in cell extracts of E. COLI: By testing heteroduplex DNA (34mers) containing C/C mismatches, two specific band shifts were generated in the gels. The band shifts were due to mismatch-specific binding of proteins present in the extracts. Cell extracts of a mutant strain defective in MutM protein did not produce a low-mobility complex. Purified MutM protein bound efficiently to the C/C mismatch-containing heteroduplex to produce the low-mobility complex. The second protein, which produced a high-mobility complex with the C/C mismatches, was purified to homogeneity, and the amino acid sequence revealed that this protein was the FabA protein of E.COLI: The high-mobility complex was not formed in cell extracts of a fabA mutant. From these results it is possible that MutM and FabA proteins are components of repair pathways for C/C mismatches in E.COLI: Furthermore, we found that Saccharomyces cerevisiae OGG1 protein, a functional homolog of E.COLI: MutM protein, could specifically bind to the C/C mismatches in DNA.

Histone-like protein HU is required for recA gene-dependent DNA repair and SOS induction pathways in UV-irradiated Escherichia coli.

PURPOSE: Escherichia coli HU protein exists as a heterodimer composed of two highly homologous subunits, HU-1 and HU-2, encoded by the hupB and hupA genes, respectively. It introduces negative supercoils into a relaxed circular DNA. Various roles of HU have been suggested in cellular processes such as DNA replication and transcription. The present experiments were designed to understand the role of HU in DNA repair processes in E. coli. MATERIALS AND METHODS: The sensitivity of hupA/hupB mutants of E. coli to the lethal and mutagenic effects of UV was compared with that of a wild-type strain. The effect of the hupAhupB mutations in SOS induction was also examined. RESULTS: The hupAhupB mutations increased the UV sensitivity of E. coli. Nucleotide excision repair was unaffected by the deficiency of HU. On the other hand, E. coli hupAhupB mutants were sensitive to UV in the recA+recB+recF background but not in the recArecB+recF+ or recA+recBrecF+ background. The frequency of UV-induced mutation to rifampicin resistance was significantly reduced in the hupAhupB mutants, and the induction of the recA::lacZ and umuC::lacZ fusion genes was also suppressed in the mutants. CONCLUSIONS: HU protein plays a critical role in the recA, recB-dependent recombinational DNA repair and SOS induction pathways in UV-irradiated E. coli.

Differential subcellular localization of human MutY homolog (hMYH) and the functional activity of adenine:8-oxoguanine DNA glycosylase.

The post-replicative adenine:8-oxo-7,8-dihydroguanine (GO) mismatch is crucial for G:C to T:A transversion. This mismatch is corrected by Escherichia coli MutY which excises the adenine from A:GO. A candidate gene coding for the human counterpart of MutY has been cloned as hMYH. However, the function and enzyme activities of the gene product have not been identified. We previously demonstrated that an epitope-tagged hMYH protein behaves as a mitochondrial protein. In the present study, we have identified an alternative hMYH transcript, termed type 2, which differs in the exon 1 sequence of the known transcript (type 1). A nuclear localization for the type 2 protein was revealed by detection of epitope-tagged protein in COS-7 cells. Expression of both type 1 and type 2 transcripts was reduced in post-mitotic tissues. hMYH cDNA suppressed the mutator phenotype of E.coli mutY. In vitro expressed hMYH showed adenine DNA glycosylase activity toward the A:GO substrate. The protein can bind to A:GO, and to T:GO and G:GO without apparent catalysis. These results represent the first demonstration of the function of the hMYH gene product which is differentially transported into the nucleus or the mitochondria by alternative splicing

The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity.

During embryonic development, initially similar fields can develop into distinct structures, such as the vertebrate fore- and hindlimbs. Although considerable progress has been made in our understanding of the genetic control underlying the establishment of the different limb axes, the molecular cues that specify the differential development of the fore- and hindlimbs are unknown. Possible candidates for genes determining limb identity are Pitx1, a gene whose transcripts are detected in the early hind- but not forelimb bud, and two members of the T-box (Tbx) gene family, Tbx4 and Tbx5, which are specifically expressed in the hindlimb and forelimb buds, respectively. Here we show that Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the activity of three signalling proteins that are required for limb outgrowth and patterning: fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and Wnt. In addition, we provide evidence that Tbx4 and Tbx5 are involved in controlling limb identity. Our findings provide insight into how similar developmental fields can evolve into homologous but distinct structures.

Replication in vitro and cleavage by restriction endonuclease of 5-formyluracil- and 5-hydroxymethyluracil-containing oligonucleotides.

PURPOSE: To investigate the biological consequences of 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). MATERIALS AND METHOD: The authors constructed 22-mer oligonucleotides containing a 5-foU or 5-hmU residue at the same sites. The effects of such modifications on the ability to serve as a template for DNA polymerase and on the cleavage by sequence-specific restriction endonuclease were examined. RESULTS: The Klenow fragment of DNA polymerase I and Thermus thermophilus DNA polymerase read through the sites of 5-foU and 5-hmU in the templates. 5-FoU directed the incorporation of dCMP in addition to dAMP opposite the lesion during DNA synthesis. The DNA polymerases incorporated only dAMP opposite the 5-hmU. The substitution of thymine by 5-foU within the recognition site of the restriction endonucleases HincII and SalI inhibited or prevented the cleavage by the enzymes, whereas the enzymes cleaved the 5-hmU-containing oligonucleotides at the same rate as the T-containing oligonucleotides. CONCLUSIONS: These results indicated that the 5-foU-A base pair is less stable than the T-A base pair and that 5-foU can form a base pair with C in addition to A. It was also demonstrated that the oxidation of thymine to 5-hmU does not result in substantial deterioration.

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions.

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.

3'-blocking damage of DNA as a mutagenic lesion caused by hydrogen peroxide in Escherichia coli.

Ionizing radiation and hydrogen peroxide (H2O2) produce many types of oxidative DNA damage such as strand breaks, apurinic/apyrimidinic (AP) sites, base modifications and 3'-blocking damage such as 3'-phosphoglycolated and 3'-phosphorylated termini. AP sites and 3'-blocking damage are repairable by exonuclease III and endonuclease IV in Escherichia coli. XthA-nfo double mutants of E. coli, which are deficient in exonuclease III and endonuclease IV, were highly sensitive to lethal and mutagenic effects of H2O2, compared with the wild-type strains. The pNT180 and pNT186 plasmids containing wild-type nfo and mutant nfo-186 gene, respectively, were introduced into the xthA-nfo mutant. The nfo-186 gene product, Nfo186, retained normal AP endonuclease activity but could not remove 3'-blocking damage from DNA. The pNT180 corrected the sensitivity of the xthA-nfo mutant to lethal and mutagenic effects of H2O2. On the other hand, the pNT186 did not have any complementation effects. From these results it was concluded that 3'-blocking damage rather than an AP site is the primary lesion responsible for both lethal and mutagenic effects of H2O2.

Different mechanisms of thioredoxin in its reduced and oxidized forms in defense against hydrogen peroxide in Escherichia coli.

The present experiments were done to elucidate the roles of thioredoxin and thioredoxin reductase system in defense against hydrogen peroxide (H2O2) in Escherichia coli. The thioredoxin-deficient mutant (trxA) was more sensitive to H2O2 than was the wild-type strain, when challenged in the stationary and exponentially growing phase. Thioredoxin reductase-deficient mutant (trxB) in the stationary phase also exhibited increased sensitivity, compared with the wild-type strain. These results indicated that reduced form of thioredoxin is required for defense against H2O2, possibly by scavenging radicals generated in the cells. In contrast, the trxB mutant in the growing phase had higher survival after exposure to H2O2 than the wild-type strain. The acquirement of resistance related to increased capacity for removing H2O2 in the trxB mutant and was not observed in a catalase-negative background. Furthermore, enhanced expression of the katG :: lacZ gene occurred in the mutant. Therefore, it was concluded that oxidized form of thioredoxin confers H2O2 resistance on E. coli cells by increasing activity to remove H2O2, which was brought about by enhanced induction of the katG-coded catalase/hydroperoxidase I at the transcriptional level. In addition, this resistance to H2O2 correlated well with reduced amount of DNA damage caused by H2O2, determined by the induction level of the recA :: lacZ fusion gene after treatment with H2O2.

Replication of DNA templates containing 5-formyluracil, a major oxidative lesion of thymine in DNA.

5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.

Induction of repair capacity for oxidatively damaged DNA as a component of peroxide stress response in Escherichia coli.

We examined whether or not peroxide stress induces a repair capacity for oxidatively damaged DNA in Escherichia coli cells. Peroxide stress was brought about by adding 30 microM hydrogen peroxide (H2O2) to exponentially growing cells. The following results were obtained. (1) After exposure to H2O2, E. coli resistance to X-rays was enhanced. The acquisition of resistance was inhibited by rifampicin and chloramphenicol. (2) The response was acquired in mutants defective in the katG and oxyR genes, as well as in the wild-type strain. (3) Lambda phages damaged by exposure to H2O2 showed higher survival on H2O2-treated cells than on untreated cells. (4) The peroxide stress did not render E. coli cells resistant to UV and mitomycin C. These suggest that peroxide stress induces a repair capacity against oxidative DNA damage and that this response must be regulated by a different mechanism from oxyR(+)-mediated regulatory system.

Enzymatic release of 5-formyluracil by mammalian liver extracts from DNA irradiated with ionizing radiation.

To identify a repair enzyme for 5-formyluracil (5-FU) caused by ionizing radiation in DNA, we used a radiolabelled product-release assay for this thymine-damaged substrate. Double-stranded poly(dA-dT)-poly(dA-dT) was radiolabelled by nick translation with [2-14C]-thymidine triphosphate. The DNA was irradiated with X-rays and incubated with cell extract from mouse liver. Radiolabelled products released from the irradiated DNA into an ethanol-soluble fraction were analysed by reversed-phase hplc. Released 5-FU was detected as a free base during reaction with the cell extract. 5-Formyl-2'-deoxyuridine was not detected in the ethanol supernatant. Boiling the extract at 97 degrees C for 15 min completely abolished its ability to release 5-FU. Similar enzymatic activity was observed with rat liver extract. These results demonstrated that mammalian cells have enzymatic activity to release 5-FU from DNA.