Frequency Of DNA Lesions Research Articles

Accumulation of true single strand breaks and AP sites in base excision repair deficient cells

Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells. However, the neutralization of methoxyamine is required prior to usage. Here we developed a convenient, specific SSB assay using alkaline gel electrophoresis (AGE) coupled with a neutral O-hydroxylamine, O-(tetrahydro- 2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes abasic sites (AP sites) to prevent their alkaline incision while still allowing for strong alkaline DNA denaturation. DNA from DT40 and isogenic polymerase β null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE (OTX-AGE) assay. Time-dependent increases in SSBs were detected in each cell line with more extensive SSB formation in the null cells. These findings were supported by an assay that indirectly detects SSBs through measuring NAD(P)H depletion. An ARP-slot blot assay demonstrated a significant time-dependent increase in AP sites in both cell lines by 1 mM MMS compared to control. Furthermore, the Pol β-null cells displayed greater AP site formation than the parental DT40 cells. OTX use represents a facile approach for assessing SSB formation, whose benefits can also be applied to other established SSB assays.

Replication through an abasic DNA lesion: structural basis for adenine selectivity

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A-rule. In this study, we present X-ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non-instructive lesions by DNA polymerases at least from this sequence family.

The Block of DNA Polymerase δ Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase β and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol delta, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol beta and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.

Uracil within DNA: an actor of antiviral immunity

Uracil is a natural base of RNA but may appear in DNA through two different pathways including cytosine deamination or misincorporation of deoxyuridine 5'-triphosphate nucleotide (dUTP) during DNA replication and constitutes one of the most frequent DNA lesions. In cellular organisms, such lesions are faithfully cleared out through several universal DNA repair mechanisms, thus preventing genome injury. However, several recent studies have brought some pieces of evidence that introduction of uracil bases in viral genomic DNA intermediates during genome replication might be a way of innate immune defence against some viruses. As part of countermeasures, numerous viruses have developed powerful strategies to prevent emergence of uracilated viral genomes and/or to eliminate uracils already incorporated into DNA. This review will present the current knowledge about the cellular and viral countermeasures against uracils in DNA and the implications of these uracils as weapons against viruses.

C → T mutagenesis and γ-radiation sensitivity due to deficiency in the Smug1 and Ung DNA glycosylases

The most common genetic change in aerobic organisms is a C:G to T:A mutation. C --> T transitions can arise through spontaneous hydrolytic deamination of cytosine to give a miscoding uracil residue. This is also a frequent DNA lesion induced by oxidative damage, through exposure to agents such as ionizing radiation, or from endogenous sources that are implicated in the aetiology of degenerative diseases, ageing and cancer. The Ung and Smug1 enzymes excise uracil from DNA to effect repair in mammalian cells, and gene-targeted Ung(-/-) mice exhibit a moderate increase in genome-wide spontaneous mutagenesis. Here, we report that stable siRNA-mediated silencing of Smug1 in mouse embryo fibroblasts also generates a mutator phenotype. However, an additive 10-fold increase in spontaneous C:G to T:A transitions in cells deficient in both Smug1 and Ung demonstrates that these enzymes have distinct and nonredundant roles in suppressing C --> T mutability at non-CpG sites. Such cells are also hypersensitive to ionizing radiation, and reveal a role of Smug1 in the repair of lesions generated by oxidation of cytosine.

Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time.

DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or during the base excision repair pathways. Here we established a new real-time assay to assess an imbalance of DNA SSB repair by indirectly measuring PARP-1 activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye. While this assay is not a direct method, it does not require DNA extraction or alkaline treatment, both of which could potentially cause an artifactual induction of SSBs. In addition, it takes only 4 h and requires less than a half million cells to perform this measurement. Using this assay, we demonstrated that the dose- and time-dependent depletion of NAD(P)H in XRCC1-deficient CHO cells exposed to methyl methanesulfonate. This decrease was almost completely blocked by a PARP inhibitor. Furthermore, methyl methanesulfonate reduced NAD(P)H in PARP-1+/+ cells, whereas PARP-1-/- cells were more resistant to the decrease in NAD(P)H. These results indicate that the analysis of intracellular NAD(P)H level using water-soluble tetrazolium salt can assess an imbalance of SSB repair in living cells in real time.

Spatial and Temporal Cellular Responses to Single-Strand Breaks in Human Cells

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.

Spatial and temporal cellular responses to single-strand breaks in human cells.

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.

DNA Damage Induced by 4,4′-Methylenedianiline in Primary Cultures of Hepatocytes and Thyreocytes from Rats and Humans

4,4′-Methylenedianiline (MDA), an aromatic amine used in various industrial processes and previously found to induce tumor development in liver and thyroid of mice and rats, was evaluated for its DNA-damaging activity in primary cultures of hepatocytes and thyreocytes from rat and human donors. After exposure for 4 and 20 h to MDA concentrations ranging from 10 to 180 μM, a statistically significant increase in the frequency of DNA lesions was revealed by the Comet assay in primary hepatocytes and thyreocytes from donors of both species, the response being dose dependent up to 56–100 μM MDA. DNA fragmentation was more marked after 4 than after 20 h exposure in all four cell types. DNA was damaged to a lesser extent in human hepatocytes and thyreocytes than in corresponding rat cells and in both species in hepatocytes than in thyreocytes. In both rat and human hepatocytes a 20-h exposure to the same MDA concentrations elicited a modest amount of DNA repair synthesis, as evaluated by autoradiography. Evidence of a partial reduction of DNA damage, and therefore of only partial DNA repair, was observed in rat hepatocytes and in rat and human thyreocytes incubated for 16 h in MDA-free medium after a 4-h MDA treatment. A 4-h exposure to 56, 100, and 180 μM MDA did not induce DNA lesions in primary cultures of cells from three rat organs, kidney, urinary bladder mucosa, and brain, which are resistant to MDA carcinogenic activity. Under the same experimental conditions any evidence of DNA damage was absent in primary kidney and urinary bladder cells from human donors. Taken as a whole the results of this work indicate that MDA is specifically activated to DNA-damaging reactive species by hepatocytes and thyreocytes in both rats and humans and thus suggest that liver and thyroid might be the targets of the carcinogenic activity of MDA also in humans.

Involvement of mammalian OGG1(MMH) in excision of the 8-hydroxyguanine residue in DNA 1,2

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase and apurinic (AP) site lyase activity. cDNA clones of four isoforms (types 1a, 1b, 1c, and 2) of human OGG1 homologs (hMMH) were isolated. In order to examine whether expression of hMMH (hOGG1) protein actually occurs in human cells, we prepared type 1a specific antibody, and by using this antibody, we showed that type 1a protein isolated from HeLaS3 has 8-OH-G glycosylase/lyase activity. Furthermore, we showed that type 1a protein is a major enzyme for repair of the 8-OH-G lesion in human cells. In our second study, we generated a mouse line carrying an inactivated mutant Mmh allele by targeted gene disruption. Liver extracts of Mmh homozygous mutant mice were found to have loss of the nicking activity for the 8-OH-G site. In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mice increased linearly with age, reaching 7-fold increase in 14 week old mice, over that of wild-type or heterozygous mice. Furthermore, when homozygous mice were fed the oxygen radical-forming agent KBrO 3, to provide oxidative stress, the level of 8-OH-G in kidney DNA was tremendously increased: more than 200-fold as that of control mice without oxidative stress after 12 weeks of age. These results indicate that Ogg1/ Mmh plays an essential role in the repair of the 8-OH-G residue in DNA produced by oxidative stress.

Incision of AP sites in lung cancer patients: a pilot study.

DNA base excision repair (BER) removes frequent DNA lesions of either endogenous or exogenous origin. Some indications point to BER defects in lung cancer patients. We have investigated the ability of ten lung cancer patients to repair natural AP sites, the most frequent genetic lesion, using an in vitro assay in which peripheral blood lymphocytes (PBL) extracts incise randomly depurinated plasmid DNA. The median value of repair activity in patients was lower than that of matched controls but the difference did not reach significance. Unlike other BER enzymatic steps, marked defects in incision of AP sites may not be associated with lung carcinogenesis.

Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat

Auramine, a dye previously found to be a liver carcinogen in both mice and rats, was evaluated for its DNA-damaging and clastogenic activities in primary cultures of rats and human hepatocytes and for the induction of DNA single-strand breaks in the liver and urinary bladder mucosa of intact rats. A similar dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic concentrations ranging from 10 to 32 μM. In contrast, neither rat nor human hepatocytes displayed an increased frequency of micronuclei after a 48-h exposure to the same auramine concentrations. In rats given a single oral dose of 125, 250 or 500 mg kg −1 auramine, the Comet assay revealed a significant increase in the frequency of DNA lesions in the liver and in the urinary bladder mucosa, the effect being slightly more marked in the liver. Taken as a whole and compared with previous findings, these results suggest that auramine is biotransformed into reactive species in target organs of both rats and humans, and that this dye might play by itself the main role in the increased incidence of bladder cancer which has been judged as causally related to its manufacture.

Search for DNA repair inhibitors: selective binding of nucleic bases-acridine conjugates to a DNA duplex containing an abasic site.

The abasic site is one of the most frequent DNA lesions generated by spontaneous or enzymatic cleavage of the N-glycosidic bond. The abasic site is also an intermediate in the nucleotide and base excision DNA repair. We examined molecules which recognize and cleave DNA at the abasic site with high efficiency. These molecules incorporate in their structure a nucleic base for abasic site recognition, an intercalator for DNA binding, and a polyamino linker for ionic interaction and DNA cleavage. Such compounds, by interfering with abasic sites in DNA, are also inhibitors of DNA repair. In order to better understand the parameters of the interaction, we carried out a UV thermal denaturation study of synthetic oligonucleotides containing the lesion both in the absence and in the presence of the drugs. A similar study was also carried out using the corresponding nonmodified oligonucleotide. The results indicate selective binding of the base-chain-intercalator conjugates to the abasic site containing oligonucleotides.

Comparison of DNA alkylation, fragmentation, and repair in maternal and fetal tissues of pregnant rats treated with a single dose of ethyl methanesulfonate, ethyl‐N‐nitrosourea, N‐nitrosodiethylamine, and methyl‐N‐nitrosourea

The occurrence and persistence of DNA damage, as detected by the alkaline elution technique, have been studied in some tissues of both fetal and adult Sprague-Dawley rats (18th day of gestation) after administration of a single equimolar dose (0.5 mmol/kg) of ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-nitrosodiethylamine (NDEA), and N-methyl-N-nitrosourea (MNU). EMS, ENU, and MNU, injected intravenously, produced a statistically significant increase of DNA elution rate, which is considered indicative of DNA fragmentation, in both maternal and fetal liver, kidney, and brain. NDEA, introduced by gastric gavage, induced DNA breaks in both liver and kidney of dams, but only in the liver of fetuses. The frequency of DNA lesions was found to vary with the four alkylating agents and in the three organs tested, to exhibit a different time course, and usually to be higher in maternal than in fetal tissues. Results provided by the concomitant determination of DNA binding levels demonstrated a satisfactory correlation with the amounts of DNA fragmentation. In contrast, the values of both these parameters did not show any positive correlation with the different susceptibility of the three organs to tumor induction. In conclusion, these findings suggest that when a compound is not available in radiolabeled form, measurement of DNA fragmentation may represent a useful alternative to the determination of DNA binding level in order to obtain information on the distribution of its reactive species in maternal and fetal tissues.

Sequence specificity in photoreaction of various psoralen derivatives with DNA: role in biological activity.

The sequence specificity in the photoreaction of various psoralen derivatives with DNA is investigated by using DNA sequencing methodology. The 3'-5' exonuclease activity associated with T4 DNA polymerase serves as a probe to map the psoralens' photoaddition (monoadducts plus biadducts) on DNA fragments of defined sequence. This approach has already allowed us to demonstrate a strong sequence context effect on the 8-methoxypsoralen photobinding to DNA [Sage, E., & Moustacchi, E. (1987) Biochemistry 26, 3307-3314]. The psoralens studied include bifunctional derivatives [8-methoxypsoralen, 5-methoxypsoralen, and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen] and monofunctional derivatives (angelicin, 3-carbethoxypsoralen, and three pyridopsoralens). Maps of photochemical binding on two DNA fragments of the lacI gene of Escherichia coli are established for all the derivatives. These maps demonstrate the following general qualitative rules in the photoreaction of the furocoumarins with DNA: thymine residues in a GC environment are cold, adjacent thymines are better targets, 5'-TpA sites are strongly preferred versus 5'-ApT, and alternating (AT)n sequences are hot spots for photoaddition. Depending on the chemical structure of the derivatives and on their affinity for DNA, some minor differences in the binding spectrum are detected. A most interesting example is 3-carbethoxypsoralen, which specifically reacts with (AT)n sites. Our observations lead us to define two types of target sites: the "strong sites", which are preferential targets for all psoralen derivatives, and the "weak sites", which are targets only for derivatives having a high affinity for DNA. The frequency of DNA lesions is much higher in the former sites.(ABSTRACT TRUNCATED AT 250 WORDS)