A study of the effect of caffeine upon excision repair of damaged DNA.

Cultured mammalian cells incur damage to their DNA when exposed to ultraviolet light or adduct-producing mutagens such as 4-nitroquinoline-1-oxide (4NQO). At least two processes are important in repair of such damage: post-replication repair and excision repair. Many researchers have reported that caffeine inhibits the former process, which occurs in connection with semiconservative DNA replication, especially in rodent cell lines such as mouse lymphoma or Chinese hamster. Excision repair is not generally considered caffeine-sensitive, although the data are somewhat conflicting because some studies had used rodent cells, which show little or no excision repair, or human cells in which alternate repair processes may have been operating.Human peripherhal blood lymphocytes from healthy donors were treated with UV light or 4NQO in order to produce pyrimidine dimers or adducts. Caffeine at concentrations of 0.75–3.0 mM was included in some cultures. The cells treated with caffeine were incubated for 90 min prior to mutagen treatment and for the entire period thereafter until cell harvests. [³H]Thymidine was added and the uptake quantitated as a measure of DNA repair. DNA replication was inhibited by hydroxyurea, so that only excision repair was measured by this method. Separate plates of cells not exposed to mutagens exhibited negligible or low thymidine uptakes.Following harvest, the cells were lysed and the DNA extracted. The DNA released was measured spectrophotometrically and then placed into liquid-scintillation counter (LSC) vials for measurement of incorporated radioactivity. Resulting cpm/μ DNA were compared for cells with and without caffeine. Lymphocytes from patients with systemic lupus erythematosus (SLE), who previously had demonstrated reduced levels of excision repair under these conditions, were also tested with caffeine. Caffeine did not inhibit repair by normal lymphocytes and the reduced repair seen in the SLE patients was not further reduced in its presence.In a series of pulse-chase experiments, some cells were treated with 4NQO and allowed to incubate with [³H]thymidine for 3 h and were harvested at the end of this period, while others were given a 13-h chase i n cold thymidine before harvest. The cpm/μg DNA for both groups were virtually identical, both in the presence and absence of 2.0 mM caffeine.     

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate in Escherichia coli and in V-79 chinese hamster cells

Caffeine increases the number of sister-chromatid exchanges (SCE) induced by mitomycin C (MMC) in human peripheral lymphocytes in culture. This enhancement decreases when the treated cells are held in medium before phytohemagglutinin (PHA) stimulation, or when caffeine is added to cultures some time after PHA stimulation but prior to DNA synthesis. There thus appears to be a caffeine-sensitive prereplication repair system, presumably an excision mechanism, capable of repairing a fraction of the MMC-induced DNA lesions.     

Exonuclease proofreading by human mitochondrial DNA polymerase

We have examined the ability of the human mitochondrial DNA polymerase to correct errors in DNA sequence using single turnover kinetic methods. The rate of excision of single-stranded DNA ranged from 0.07 to 0.17 x s(-1), depending on the identity of the 3'-base. Excision of the 3'-terminal base from correctly base paired DNA occurred at a rate of 0.05 x s(-1), indicating that the cost of proofreading is minimal, as defined by the ratio of the k(exo) for correctly base-paired DNA divided by the rate of forward polymerization (0.05/37 = 0.14%). Excision of duplex DNA containing 1-7 mismatches was biphasic, and the rate and amplitude of the fast phase increased with the number of mismatches, reaching a maximum of 9 x s(-1). We showed that transfer of DNA from the polymerase to the exonuclease active site and back again occurs through an intramolecular reaction, allowing for a complete cycle of reactions for error correction. For DNA containing a buried mismatch (T:T followed by C:G base pairs), the 3' base was removed at a rate of 3 x s(-1). The addition of nucleotide to the reaction that is identical to the 3' base increased the rate of excision 7-fold to 21 x s(-1). We propose that the free nucleotide enhances the rate of transfer of the DNA to the exonuclease active site by interrupting the correct 3' base pair through interaction with the template base. The exonuclease contribution to fidelity is minimal if the calculation is based on hydrolysis of a single mismatch: (k(exo) + k(pol,over))/(k(pol,over)) = 10, but this value increases to approximately 200 when examining error correction in the presence of nucleotides.     

Caffeine-inhibited DNA repair in 7-bromomethylbenz (a)-anthracene-treated Chinease hamster cells: formation of breaks in parental DNA and inhibition of ligation of nascent DNA as a mechanism for enhancement of lethality and chromosome damage

The cytotoxic and clastogenic effects of 7-bromomethylbenz(a)anthracene (7-BMBA) are potentiated by post-treatment incubation of cells in the presence of an non-toxic concentration of caffeine. Under these conditions caffeine inhibits the rate of ligation of newly-synthesised DNA and induces breaks in the template strand of DNA. It is proposed that endonucleolytic attack occurs at the site of lesions in the template strand of DNA and that a later step(s) of excision-repair is (are) inhibited by the presence of caffeine-induced 'gap' in the nascent DNA opposite these lesions.     

Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant.

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.     

Repair of the major lesion resulting from C5'-oxidation of DNA

Oxidation of the C5'-position of DNA results in direct strand scission. The 3'-fragments produced contain DNA lesions at their 5'-termini. The major DNA lesion contains an aldehyde at its C5'-position, but its nucleobase is unmodified. Excision of the lesion formed from oxidation of thymidine (T-al) is achieved by strand displacement synthesis by DNA polymerase β (Pol β) in the presence or absence of flap endonuclease 1 (FEN1). Pol β displaces T-al and thymidine with comparable efficiency, but less so than a chemically stabilized abasic site analogue (F). FEN1 cleaves the flaps produced during strand displacement synthesis that are two nucleotides or longer. A ternary complex containing T-al is also a substrate for the bacterial UvrABC nucleotide excision repair system. The sites of strand scission are identical in ternary complexes containing T-al, thymidine, or F. UvrABC incision efficiency of these ternary complexes is comparable as well but significantly slower than a duplex substrate containing a bulky substituted thymidine. However, cleavage occurs only on the 5'-fragment and does not remove the lesion. These data suggest that unlike many lesions the redundant nature of base excision and nucleotide excision repair systems does not provide a means for removing the major damage product produced by agents that oxidize the C5'-position. This may contribute to the high cytotoxicity of drugs that oxidize the C5'-position in DNA.     

The efficiency of hypoxanthine excision by alkyladenine DNA glycosylase is altered by changes in nearest neighbor bases

Alkyladenine DNA glycosylase (AAG) excises a structurally diverse group of damaged purines including hypoxanthine, 1,N(6)-ethenoadenine, 3-methyladenine, and 7-methylguanine from DNA to initiate base excision repair at these sites. Excision occurs in an enzyme.DNA complex in which the damaged base is flipped out of the DNA helix into the enzyme active site. To determine whether local DNA sequence could affect the overall efficiency of excision of hypoxanthine from DNA, single-turnover kinetics of excision, AAG.DNA binding, and melting temperatures were measured for DNA substrates that differed in the base pairs immediately 5' and 3' to hypoxanthine. When Hx was flanked by a 5'G and a 3'C, the efficiency of excision was reduced dramatically in comparison to a duplex containing a 5'T and 3'A. The reduction in excision efficiency was largely due to a decrease in binding affinity of AAG for DNA. The overall effect of GC versus TA nearest neighbors was to magnify the difference in the efficiencies of excision of Hx from pairs with thymine and difluorotoluene from a factor of 5 to a factor of about 100. In general, DNA substrates that were more stable as indicated by higher melting temperatures gave reduced efficiencies of excision of Hx. These results are discussed in terms of a model in which the relative stabilities of base-flipped versus unflipped complexes contribute the overall efficiency of excision and substrate specificity of AAG.     

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.     

Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein.

The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated. DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA. Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. Kinetic studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde). Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyuracil, were not excised. Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature. The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage. Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate. These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.     

Lambda excision revisited: testing a model for synapsis of prophage ends

Excision of lambda prophage was reexamined to test a model for prophage end synapsis. The model proposes that, during in situ prophage replication, following induction, the diverging replication forks are held together. Consequently, prophage DNA is spooled through the replication machinery, drawing the prophage ends together and facilitating synapsis. The model predicts that excision will be slowed if in situ lambda replication is inhibited, and the predicted low rate of excision of a nonreplicating prophage was observed after thermoinduction. However, excision was rapid if additional Int protein was supplied or if the temperature was reduced after induction, showing that (i) Int is partially thermosensitive for excision at 42 degrees C and (ii) in situ replication is not required for rapid excision, a finding that is inconsistent with the model.     

USP7/HAUSP stimulates repair of oxidative DNA lesions

USP7 is involved in the cellular stress response by regulating Mdm2 and p53 protein levels following severe DNA damage. In addition to this, USP7 may also play a role in chromatin remodelling by direct deubiquitylation of histones, as well as indirectly by regulating the cellular levels of E3 ubiquitin ligases involved in histone ubiquitylation. Here, we provide new evidence that USP7 modulated chromatin remodelling is important for base excision repair of oxidative lesions. We show that transient USP7 siRNA knockdown did not change the levels or activity of base excision repair enzymes, but significantly reduced chromatin DNA accessibility and consequently the rate of repair of oxidative lesions.     

Repair of oxidative damage of thymine by HeLa whole-cell extracts: simultaneous analysis using a microsupport and comparison with traditional PAGE analysis

In mammalian cells, the base excision repair (BER) pathway allows the remove of small DNA base lesions such as oxidized bases. It is initiated by glycosylases that removed the modified base leaving an abasic site that is subsequently processed by AP endonuclease activities. Measurement of BER activities in cell extracts is time consuming and hazardous when radioactive material is used. We report in this study, the parallelized fluorescent analysis of excision of several oxidation products of thymine by cell extracts. To conduct the study, 5-(hydroxymethyl)uracil, 5-formyluracil, 5-carboxyuracil and formylamine together with uracil and the control thymine, were incorporated into oligonucleotides of identical sequences and paired either with adenine or with guanine containing DNA fragments. The oligonucleotides were fixed by sandwich hybridization in wells of a microplate (OLISA technology). Excision by HeLa extracts of the six different DNA base lesions could be followed simultaneously in the same well. Our results showed that the extent of excision of the lesions was the same on support and in solution using classical PAGE analysis approach with modified (32)P-labeled oligonucleotides. We demonstrated that the simultaneous analysis on support is a successful approach to facilitate high-throughput screening of BER activities present in cell extracts. Moreover, extended study of 5-carboxyuracil revealed that this lesion displays similar biological properties as 5-formyluracil.     

Characterization of Excision Repair in Neurospora crassa

The excision of pyrimidine dimers from the deoxyribonucleic acid (DNA) of Neurospora crassa was examined. Postirradiation incubation in the presence of several chemicals known to inhibit various repair systems indicated that caffeine reduced the rate of excision twofold, but did not inhibit excision completely as did proflavine and quinacrine. Examination of the time course of excision showed that repair occurs at a relatively rapid rate: approximately 60 dimers excised per min after 500 ergs/mm(2). Further evidence for rapid excision was obtained by sedimentation analysis of DNA; the maximal number of breaks introduced during repair was three, suggesting that breaks are repaired almost as fast as they are made and that only a few dimers are repaired at a time. Repair synthesis was measured by prelabeling the DNA with (15)N and D(2)O, and then subjecting the DNA to equilibrium density gradient centrifugation after postirradiation incubation with (32)P. Accumulation of single-strand breaks with increasing dose of ultraviolet radiation suggested that the limiting step was subsequent to the incision and excision steps of repair. Equilibrium CsCl centrifugation demonstrated that the limiting step in excision was repair synthesis.     

Nucleotide excision repair from site-specifically platinum-modified nucleosomes

Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum-based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a site-specific cis-diammineplatinum(II)-DNA intrastrand d(GpG) or a d(GpTpG) cross-link. The ability of the histone core to modulate the excision of these defined platinum adducts was investigated as a model for exploring the cellular response to platinum-DNA adducts in chromatin. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. With the GTG-Pt DNA substrate, the nucleosome inhibits excision to about 10% of the level observed with free DNA, whereas with the less efficient GG-Pt DNA substrate the nucleosome inhibited excision to about 30% of the level observed with free DNA. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is approximately 2-fold higher than the level observed with recombinant material. This result reveals that post-translational modification of histones can modulate nucleotide excision repair from damaged chromatin. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

Repair of UV-irradiated plasmid DNA in Saccharomyces cerevisiae. Inability to complement mutational defects in excision repair by in vitro treatment with Micrococcus luteus UV endonuclease

Excision repair defects of Saccharomyces cerevisiae rad1-1, rad4-4, rad7-1 and rad14 mutants were examined. As previously found, transformation of such cells with UV-irradiated plasmid DNA is poor compared to wild-type yeast. Treatment of UV-irradiated YRp12 plasmid DNA with crude preparations of Micrococcus luteus UV endonuclease before introducing it into rad1-1 cells increased transformation efficiency to wild-type levels. This is consistent with earlier reports of rad1-1 mutants being defective in the incision step of excision repair. However, with purified UV endonuclease little or no rescue occurred when the UV-irradiated plasmid was incised before transformation into rad1-1 or rad4-4 cells. Furthermore, the purified UV endonuclease reduced transformation of rad7-1 and rad14 mutants to levels seen in rad1-1 and rad4-4 cells. In contrast such treatment caused only a small decrease in the transforming ability of UV-irradiated DNA in wild-type cells. These results show that yeast can normally process pre-incised, UV-irradiated DNA and that this activity is absent in rad1-1, rad4-4, rad7-1 and rad14 mutants. Thus, in addition to their previously reported roles in incision, the RAD1, 4, 7 and 14 gene products are also required for repair to continue after the incision of DNA lesions.     

Transformation of ultraviolet-irradiated human fibroblasts by simian virus 40 is enhanced by cellular DNA repair functions

Human fibroblasts irradiated with ultraviolet light were either tested for survival (colony formation) or infected with simian virus 40 and examined for transformation (foci formation). For normal cell cultures, the fractions of surviving colonies which were also transformed increased with increasing irradiation dose. In contrast, little increase in the transformation of ultraviolet-irradiated repair-deficient (xeroderma pigmentosum and xeroderma pigmentosum variant) cells was observed. Similar experiments with xeroderma pigmentosum variant cells treated with caffeine following irradiation indicated that, under these conditions, the deficient cells produced more transformants among the survivors of ultraviolet irradiation than did unirradiated cells. These results suggest (1) that DNA repair functions, not DNA damage per se, are required for enhanced viral transformation in normal cells; (2) that functions involved in excision repair and functions needed for replication of ultraviolet-damaged DNA appear necessary for this stimulation; and (3) that blocking DNA replication in ultraviolet-irradiated xeroderma pigmentosum variant cells by caffeine enhances viral transformation.     

Molecular mechanisms of stress-induced hereditary variability

The molecular mechanisms of generation of stress-induced genetic recombinations and point mutations are considered. Due to the oxidative, temperature, radiation and other forms of stress, intensive modification of DNA bases occurs. Excision of the modified bases (hypoxanthine, uracil, pyrimidine photoproducts, methylated purines) leads to the formation of single-stranded gaps in DNA. If one DNA strand is damaged, there is high probability of its primary structure being completely restored. When the rate of lesions increases, the DNA can be damaged in the gap-related opposite sites of both strands. It is shown that in this case, the excision repair leads to a burst of recombinations and point mutations which may be concerned with the mispairings, double-stranded breaks, induction of SOS-response. With the increase in the rate of lesions, the possibility of the damage in self-complementary DNA sequences is also enhanced. This leads to formation of hairpin structures in the single-stranded DNA stretches. It is demonstrated that in these cases the repair results in development of deletions, insertions and clusters of point mutations predetermined by the primary DNA structure. Independent means of stress-induced mutations' occurrence seem to be the transposable elements. The stress-induced outbreaks of recombinations provide conceivably new variants of genotypes to be selected for the adaptation to new extreme conditions.     

Substrate specificity of Deinococcus radiodurans Fpg protein.

A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.