Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.     

Why do O-alkylguanine and O-alkylthymine miscode? The relationship between the structure of DNA containing O-alkylguanine and O-alkylthymine and the mutagenic properties of these bases

The carcinogenic and mutagenic N-nitroso compounds produce GC to AT and TA to GC transition mutations because they alkylate O⁶ of guanine and O⁴ of thymine. It has been generally assumed that these mutations occur because O⁶-alkylguanine forms a stable mispair with thymine and O⁴-alkylthymine forms a mispair with guanine. Recent studies have shown that this view is mistaken and that the alkylG·T and alkylT·G mispairs are not more stable than their alkylG·C or alkylT·A counterparts. Two possible explanations based on recent structural studies are put forward to account for the miscoding. The first possibility is that the DNA polymerase might mistake O⁶-alkylguanine for adenine, and O⁴-alkylthymine for cytosine, because of the physical similarity of these bases. O⁶-Methylguanine and adenine are similarly lipophilic and X-ray crystallography of the nucleosides has shown a close similarity in bond angles and lengths between O⁶-methylguanine and adenine, and between O⁴-methylthymine and cytosine. The second possible explanation is that the important factor in the miscoding is that the alkylG·T and alkylT·G mispairs retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine while the alkylG·C and alkylT·A pairs adopt a wobble conformation. ³¹P NMR of DNA duplexes show that the phosphodiester links both 3′ and 5′ to the C have to be distorted to accomodate the O⁶-ethylguanine:C pair, whereas there is less distortion of the phosphodiesters 3′ and 5′ to the T in an ethylG·T pair. Recent kinetic measurements show that the essential aspect of base selection in DNA synthesis is the ease of formation of the phosphodiester links on both the 3′ and 5′ side of the incoming base. The Watson-Crick alignment of the alkylG·T and alkylT·G mispairs may facilitate formation of these phosphodiester links, and this alignment rather than the strength of the base pairs and the extent of hydrogen bonding between them may be the crucial factor in the miscoding. If either hypothesis is correct it suggests that previously too much emphasis has been placed on the stability of the normal pairs in the replication of DNA.     

Chromosomal instability, reproductive cell death and apoptosis induced by O-methylguanine in Mex, Mex and methylation-tolerant mismatch repair compromised cells: facts and models

O⁶-Methylguanine (O⁶-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O⁶-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O⁶-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex⁻), expressing MGMT (Mex⁺) or exhibiting the tolerance phenotype (Mex⁻, methylation resistant) were compared as to their clastogenic response. Mex⁻ cells were more sensitive than Mex⁺ cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex⁺ as compared to Mex⁻ cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex⁻ cells whereas it dropped to nearly control level in Mex⁺ cells. From this we conclude that, in the first post-treatment replication cycle of Mex⁻ cells, only a minor part of aberrations (<40%) was due to O⁶-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (>90%) was caused by the lesion. Thus, O⁶-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex⁻ cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O⁶-MeG. In methylation tolerant cells, which are characterized by impaired G–T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O⁶-MeG–T mispairs is decisively involved in O⁶-MeG born chromosomal instability and recombination. We also show that Mex⁺ and methylation tolerant cells are more resistant than Mex⁻ cells with regard to induction of apoptosis, indicating O⁶-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.     

Properties of mer HeLa cells sensitive or resistant to the cytotoxic effects of MNU; effects on DNA synthesis and of post treatment with caffeine

A line of HeLa cells was shown to be particularly sensitive to N-methyl-N-nitrosourea (MNU) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but not to variety of other cytotoxic agents. A resistant line (designated HeLa/A22), was derived by treating Hela cells repeatedly with MNU. Both the sensitive (HeLa) and resistant (Hela/A22) cells have a mer⁻ phenotype based both on their reduced rates of loss of O⁶-methylguanine (O⁶-MeG) from DNA and their low levels of the enzyme O⁶-methylguanine methyltransferase (MT). HeLa cells are therfore sensitive to unrepaired O⁶-MeG in DNA while the Hela/A22 cells are resistant to unexcised O⁶-MeG and thus the A22 cells have the mer⁻ rem⁺ phentype. MNU produced an imediate dose-dependent inhibition of DNA synthesis in cultures of both sensitive resistant cells which increased with time until about 4 h after treatment. DNA synthesis then recovered to near control rates in both sensitive and resistant cells before then exhibiting a progressive decrease after 24 h. DNA synthesis was more depressed at these late times after treatment in cultures of sensitive cells than in those of similarly-treated resistant cells. DNA synthesis remained depressed in sensitive cells but recovered 3 days after treatment in resistant cells.

Post treatment of incubation of MNU-treated HeLa cells with caffeine did not increase the toxic action of MNU. In contrast, post treatment of the resistant HeLa/A22 cells with caffeine resulted in a dramatic increase in the toxic effects of a higher equitoxic dose of MNU. The depressed rate of DNA synthesis observed in both cell lines after doses of MNU was partially reversed by post treatment with caffeine in both sensitive and resistant cells. These observations can be interpreted in terms of the effects of caffeine on DNA replication in treated cells.     

Potent induction of the adaptive response by a weak mutagen, methyl iodide, in Escherichia coli

Methyl iodide (MeI), a weakly mutagenic and highly chemoselective chemicals, was tested for its abilities to induced the adaptive and SOS responses in E. coli CSH26/pMCP1000 (alkA′-lacZ′) and CSH26/psK1002 (umuC′-lacZ′). MeI induced the adaptive response effectively but gave a very weak SOS response. Its potent ability in inducing the adaptive response was also demonstrated by adaptation to both the mutagenic and killing effects of N-methyl-N-nitrosourea (MNU) in E. coli WP2 cells. Simultaneous treatment with MeI in a non-growth medium slightly increased the mutagenicity of MNU, probably as a result of depletion of the repair enzyme, O⁶-methylguanine-DNA methyltransferase, which is constitutively present in the cells. As MeI itself proved to be only weakly mutagenic, a small part of the adaptive response which we have observed may involve indirect methylation of the repair enzyme by methyl transfer from MeI-induced O⁶-methylguanine residues in DNA. But the extent of the induced adaptive response seems to be much higher than would be expected from the observed weak mutagenicity of MeI. It is therefore suggested that the mechanism of induction of the adaptive response may involve direct methylation of the O⁶-methylguanine-DNA methyltransferase itself.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Structure of the serine-containing capsular poly-saccharide K40 antigen from Escherichia coli O8:K40:H9

The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E. coli O8:K40:H9 was elucidated by determination of the composition, ¹H- and ¹³C-n.m.r. spectroscopy, periodate oxidation and Smith degradation, and methylation analysis. The K40 polysaccharide consists of [(O-β- -glucopyranosyluronic acid)-(1→4)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→6)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→4)] repeating units. All of the glucuronic acid residues are substituted amidically with -serine.     

Induction of rat liver O6-alkylguanine-DNA alkyltransferase by bleomycin

Alkyl adducts at the O⁶-position of guanine constitute promutagenic DNA lesions likely to be involved in the initiation of malignant transformation. They can be removed by a cellular acceptor protein termed O⁶-alkylguanine-DNA alkyltransferase (AT). In rat liver this repair enzyme can be induced by a variety of hepatotoxins, partial hepatectomy and X-irradiation. This paper describes a stimulation of the hepatic AT by treatment of rats with the radiomimetic agent, bleomycin. Induction of AT is dose-dependent up to 20 mg bleomycin/kg and appears to level off with higher doses. Enhancement of O⁶-meG repair is detectable within 24 h after a single i.p. injection. Maximum AT induction was reached after 6 days and amounted to 350% of the control levels. The enhancement of AT activity is not associated with acute liver injury and initially coincides with an inhibition of [³H]deoxythymidine incorporation into hepatic DNA. This indicates that AT induction in rat liver is not necessarily dependent on tissue necrosis with increased cell replication. Since bleomycin does not produce DNA lesions recognized and repaired by the AT, the hypothesis is entertained that AT induction by these agents is part of a concerted reaction to radiation-type DNA damage.     

Binding of MutS mismatch repair protein to DNA containing UV photoproducts, "mismatched" opposite Watson--Crick and novel nucleotides, in different DNA sequence contexts

Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins-bacterial MutS, eukaryotic MSH2.MSH6 (MutSalpha) and MSH2.MSH3-initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of "mismatched" T[CPD]T/AG and T[6--4]T/AG photoproducts, reported previously for hMutSalpha and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6--4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K(1/2) 2.1--2.9 nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6--4]T/AG a forked H-bond between O2 of the 3' thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6--4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSalpha) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis.     

Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate

The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O⁶-methyl-2′-deoxyguanosine (O⁶-mdGuo) by LC–MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10⁹ nucleotides while O⁶-mdGuo was almost 50-fold lower (32 adducts per 10⁹ nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the “doubling dose”, i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7 μM MMS were deduced for 7-mG and O⁶-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66 μM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose–response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose–response relationships and for the setting of tolerable exposure levels are discussed.     

Repair of 8-oxoguanine in Saccharomyces cerevisiae: interplay of DNA repair and replication mechanisms

8-Oxo-7,8-dihydroguanine (8-oxoG) is produced abundantly in DNA exposed to free radicals and reactive oxygen species. The biological relevance of 8-oxoG has been unveiled by the study of two mutator genes in Escherichia coli, fpg, and mutY. Both genes code for DNA N-glycosylases that cooperate to prevent the mutagenic effects of 8-oxoG in DNA. In Saccharomyces cerevisiae, the OGG1 gene encodes a DNA N-glycosylase/AP lyase, which is the functional homologue of the bacterial fpg gene product. The inactivation of OGG1 in yeast creates a mutator phenotype that is specific for the generation of GC to TA transversions. In yeast, nucleotide excision repair (NER) also contributes to the release of 8-oxoG in damaged DNA. Furthermore, mismatch repair (MMR) mediated by MSH2/MSH6/MLH1 plays a major role in the prevention of the mutagenic effect of 8-oxoG. Indeed, MMR acts as the functional homologue of the MutY protein of E. coli, excising the adenine incorporated opposite 8-oxoG. Finally, the efficient and accurate replication of 8-oxoG by the yeast DNA polymerase η also prevents 8-oxoG-induced mutagenesis. The aim of this review is to summarize recent literature dealing with the replication and repair of 8-oxoG in Saccharomyces cerevisiae, which can be used as a paradigm for DNA repair in eukaryotes.     

Mutagenesis by N-nitroso compounds: relationships to DNA adducts, DNA repair, and mutational efficiencies

The relationships between DNA alkylation, DNA repair and mutagenesis by N-nitroso compounds in Salmonella were examined. DNA adducts formed by treatment of the bacteria with N-nitroso compounds were monitored. Critical to the study was establishing which adducts led to mutations. Two methods were employed. In one, correlations in the dose-responses for adducts and mutagenesis were sought. For instance O⁶-methyl- and -ethyl-guanine, in contrast to other adducts, exhibited thresholds in their accumulation in Salmonella DNA, and mutagenesis at GC base pairs also exhibited the same threshold, suggesting a dependence of mutagenesis on the O⁶-alkyguanines. In the second method, mutagenesis induced by different mutagens with overlapping adduct spectra was compared. For example, EMS and ENU generate similar ratios of adenine adducts, but only ENU produces thymine adducts, and only ENU induced AT-GC and AT-CG base changes. These observations suggested that ethylthymines led to these mutations. Furthermore, it was found that these mutations were largely dependent on the presence of the plasmid, pKM101, indicating that error-prone repair activity contributes importantly in their processing to mutations. When DNA adducts by N-nitrosopyrrolidine were examined it was found that only one major adduct was detected in an excision-repair-deficient strain, and that this adduct was not present in a repair-proficient strain. Mutagenesis was also greatly reduced in the proficient strain, suggesting that mutagenesis was dependent on this adduct. From the relationships between premutagenic adducts levels adducts. This calculation assumed an average distribution of adducts and mutations and required knowledge of the target size and the types of mutations that could lead to phenotypic changes. For the unrepaired O⁶-methyl- and -ethyl-guanines, and the O-ethylthymines the mutational efficiencies were high (ca. 30–70%), but for the N-nitrosopyrrolidine adduct it was low (ca. 1%). Initial studies were carried out on the mutational specificities of two higher homologue N-nitroso compounds (the N-nitroso-N-propyl- and N-butyl-nitroguanidines) in uvrB/pKM101 strains. This class of nitroso compounds is known to form similar DNA adducts as ENU. Their specificities were similar to that of N-nitroso-N-ethylurea at a high dose except the fraction of mutations at AT base pairs was reduced. The fraction of GC-CG transversions was although low, increased. The mutational specificities of N-nitroso-N-methylurea and N-nitrosopyrrolidine were significantly different from the specificity of ENU as would be expected from their different adduct distributions.     

Quantitative comparison of carcinogenicity, mutagenicity and electrophilicity of 10 direct-acting alkylating agents and of the initial O6:7-alkylguanine ratio in DNA with carcinogenic potency in rodents

The quantitative relationship between carcinogenicity in rodents and mutagenicity in Salmonella typhimurium was examined, by using 10 monofunctional alkylating agents, including N-nitrosamides, alkyl methanesulfonates, epoxides, β-propiolactone and 1,3-propane sultone. The compounds were assayed for mutagenicity in two S. typhimurium strains (TA1535 and TA100) and in plate and liquid assays. The mutagenic activity of the agents was compared with their alkylating activity towards 4-(4′-nitrobenzyl)pyridine and with their half-lives (solvolysis constants) in an aqueous medium. No correlations between these variables were found, nor was mutagenic activity correlated with estimates of carcinogenicity in rodents.

There was a positive relationship between carcinogenicity and the initial ratios of 7-: O⁶-alkylguanine formed or expected after their reaction with double-stranded DNA in vitro. The results suggest that alkylation of guanine at position O⁶ (or at other O atoms of DNA bases) may be a critical DNA-base modification that determines the overall carcinogenicity of these alkylating agents in rodents.     

Effects of geometry on transmission and sensing potential of tapered fiber sensors

Geometry of tapered fiber sensors critically affects the response of an evanescent field sensor to cell suspensions. Single-mode fibers (nominally at 1300 nm) were tapered to symmetric or asymmetric tapers with diameters in the range of 3–20 μm, and overall lengths of 1–7 mm. Their transmission characteristics in air, water and in the presence of Escherichia coli (JM101 strain) at concentrations of 100, 1000, 7000 and 7 million cells/mL were measured in the 400–800 nm range and gave rich spectral data that lead to the following conclusions. (1) No change in transmission was observed due to E. coli with tapers that showed no relative change in transmission in water compared to air. (2) Tapers that exhibited a significant difference in transmission in water compared to air gave weak response to the presence of the E. coli. Of these, tapers with low waist diameters (6 μm) showed sensitivity to E. coli at 7000 cells/mL and higher concentration. (3) Tapers that showed modest difference in water transmission compared to air, and those that had small waist diameters gave excellent response to E. coli at 100–7000 cells/mL. In addition, mathematical modeling showed that: (1) at low wavelength (470 nm) and small waist diameter (6 μm), transmission with water in the waist region is higher than in air. (2) Small changes in waist diameter (0.05 μm) can cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 μm. (3) For the same overall geometry, a 5.5 μm diameter taper showed larger refractive index sensitivity compared to a 6.25 μm taper at 470 nm.     

The cyclization of linear DNA in Escherichia coli by site-specific recombination

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox² plasmids) are cyclized in Cre⁺ E. coli strains after introduction either by transformation or by mini-Mu transduction, Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox² plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox² plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre⁺ E. coli recBC strains allow the efficient recovery of lox² plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.     

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS-mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.     

Reduced methylation-induced mutagenesis in rat splenocytes in vivo by sub-chronic low dose exposure to N-metyl-N-nitrosourea

Estimates of genotoxic effects of mutagens at low and protracted doses are often based on linear extrapolation of data obtained at relatively high doses. To test the validity of such an approach, a comparison was made between the mutagenicity of N-methyl-N-nitrosourea (MNU) in T-lymphocytes of the rat following two treatment protocols, i.e. sub-chronic exposure to a low dose (15–45 repeated exposures to 1 mg/kg of MNU) or acute exposure to a single high dose (15, 30 or 45 mg/kg of MNU). Mutation induction appeared dramatically lower following sub-chronic treatment compared to treatment with a single high exposure. Furthermore, DNA sequence analysis of the coding region of the hprt gene in MNU-induced mutants showed that acute high dose treatment causes mainly GC → AT base pair changes, whereas sub-chronic treatment results in a significant contribution of AT base pair changes to mutation induction. We hypothesize that O⁶-methylguanine-DNA methyltransferase is saturated after acute treatments, while after sub-chronic treatment most O⁶-methylguanine is efficiently repaired. These data suggest (i) that risk estimations at low and protracted doses of MNU on the basis of linear extrapolation of effects measured at high dose are too high and (ii) that the protective effects of DNA repair processes are relatively strong at low sub-chronic exposure.