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Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair.

The mutagenic and carcinogenic consequences of unrepaired DNA damage depend upon its precise location with respect to the relevant genomic sites. Therefore, it is important to learn the fine structure of DNA damage, in particular, proto-oncogenes, tumor-suppressor genes, and other DNA sequences implicated in tumorigenesis. Both the introduction and the repair of many types of DNA lesions are heterogeneous with respect to chromatin structure and/or gene activity. For example, cyclobutane pyrimidine dimers are removed more efficiently from the transcribed than the nontranscribed strand of the dhfr gene in Chinese hamster ovary cells. In contrast, preferential strand repair of alkali-labile sites is not found at this locus. In mouse 3T3 cells, dimers are more efficiently removed from an expressed proto-oncogene than from a silent one. Persistent damage in nontranscribed domains may account for genomic instability in those regions, particularly during cell proliferation as lesions are encountered by replication forks. The preferential repair of certain lesions in the transcribed strands of active genes results in a bias toward mutagenesis owing to persistent lesions in the nontranscribed strands. Risk assessment in environmental genetic toxicology requires assays that determine effective levels of DNA damage of producing malignancy. The existence of nonrandom repair in the mammalian genome casts doubt on the reliability of overall indicators of carcinogen-DNA binding and lesion repair for such determinations. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response to particular environmental agents.

Alkylating agent damage was quantified in human T-lymphocytes by calculating gene-specific lesion frequencies and repair rates. At 3 time points after exposure to methyl methanesulfonate (0, 6, and 24 h), T-lymphocyte DNA was extracted, digested with HindIII, and divided into 2 aliquots. Apurinic sites were formed in the DNA fragments of both aliquots by heat-induced liberation of the N-methylpurines. The methoxyamine-treated aliquot provided gene fragments which were refractory to alkaline hydrolysis (full-length fragments), while the fragments in the untreated aliquot were cleaved at apurinic sites by hydroxide. After Southern blotting, lesion frequencies were calculated by comparing the band intensity of the full-length fragment to its unprotected counterpart. The restriction fragments analyzed were from the constitutively active dihydrofolate reductase (dhfr) plus hypoxanthine phosphoribosyltransferase (hprt) genes and from the transcriptionally inactive Duchenne muscular dystrophy gene (dmd). In decreasing order, the fragments containing the most lesions per kb of DNA were: hprt greater than dhfr greater than dmd. T-Lymphocytes from 2 females had 30% more heat-labile N-methylpurines in the active X-linked hprt gene than in the inactive X-linked dmd gene. The lesion frequency found in the male's lone hprt allele was the highest observed. These lesion frequency differences are discussed in terms of chromatin structure. After 6 and 24 h, no significant repair rate differences were observed among the 3 genes.

The removal of N-methylpurines from the DHFR gene and an unexpressed adjacent locus located downstream occurs at similar rates and to a similar extent in dimethyl sulfate treated Chinese hamster ovary B11 cells. Furthermore, no significant differences in repair rates are observed between the strands of the active gene. These data primarily reflect the removal of the most abundant lesion produced by dimethyl sulfate, 7-methylguanine, and are in contrast to the results obtained for the removal of ultraviolet-induced cyclobutane pyrimidine dimers from the same region of the genome. Pyrimidine dimers are cleared preferentially from the transcribed strand of the DHFR gene and are removed poorly from the non-transcribed complementary strand and unexpressed adjacent regions. The results suggest that DNA lesions such as dimers that block transcription are removed preferentially from active genes, whereas lesions that do not interfere with nucleic acid synthesis (i.e. 7-methylguanine) are removed at similar rates from expressed and silent loci.

The activity of the DNA-repair protein O6-alkylguanine-DNA-alkyltransferase was found to be strongly inhibited by a number of metal ions. Cd2+ was the most active followed by Cu2+, Hg2+, Zn2+ and Ag2. This inhibition is likely to result from the interaction of the metals with the cysteine-acceptor residue on the protein since the inhibition was reduced by increasing the concentration of dithiothreitol in the assay buffer. These results raise the possibility that exposure to Cd2+ could increase the mutagenicity and carcinogenicity of alkylating agents by retarding the rate of repair of alkylated DNA. However, other metals or metallic compounds which are known to be carcinogenic (such as compounds containing arsenic, lead, nickel or chromium) did not interfere with DNA repair by this protein.

O6-Methylguanine in DNA is repaired by the action of a protein termed O6-alkylguanine-DNA alkyltransferase (AT) which transfers the methyl group to a cysteine residue in its own sequence. Since the cysteine which is methylated is not regenerated rapidly, if at all, the capacity for repair of O6-methylguanine is limited by the number of molecules of the AT available within the cell. The level and inducibility of the AT differed greatly in different mammalian cell types and species with the highest levels in human tissues and in liver and the lowest levels in brain. Only a small induction occurred in rat liver in response to exposure to alkylating agents. In E. coli such exposure increased the activity more than 100-fold. The At was not specific for methyl groups but also removed ethyl, 2-hydroxyethyl, n-propyl, isopropyl and n-butyl groups from the O6-position in DNA. The protein isolated from E. coli removed methyl groups much more rapidly than the larger alkyl groups but the mammalian AT isolated from rat liver showed much less difference in rate with adducts of different size. Ethyl and n-propyl groups were removed by the rat liver AT only three to four times more slowly than methyl groups. Another important difference between the bacterial and mammalian ATs is that the bacterial protein was also able to remove methyl groups from the O4-position of thymine in methylated DNA or poly(dT) but the AT from rat liver or human fibroblasts did not repair O4-methylthymidine.(ABSTRACT TRUNCATED AT 250 WORDS)

O6-Alkylguanine-DNA-alkyltransferase is a DNA repair protein known to carry out the transfer of alkyl groups from the O6-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. We have examined the ability of this protein isolated from either E. coli or mammalian cells to perform this repair reaction in short oligodeoxynucleotides. Dodecadeoxynucleotides of the sequence 5'-dCGNGAATTCm6GCG-3' where N is any one of the normal four bases were all repaired very rapidly by the protein with 50% repair in less than 15 s at 0 degree C. The hexadeoxynucleotide 5'-dCGCm6GCG-3' was repaired slightly more slowly with 50% removal taking 7 min at 0 degree C and 1.5 min at 37 degrees C. The tetradeoxynucleotide 5'-dTm6GCA-3' was also a substrate but was repaired much more slowly requiring 45 min for 50% repair at 37 degrees C. These results indicate that (a) the AGT has a strong but not absolute preference for double-stranded DNA substrates; (b) the repair of O6-methylguanine is independent of the base opposite the lesion; and (c) that oligodeoxynucleotides as short as tetramers are substrates for repair by this protein.

DNA substrates containing O6-n-butylguanine, O6-iso-butylguanine, O6-n-propylguanine and O6-iso-propylguanine were prepared by reaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea. These substrates were used to test the ability of O6-alkylguanine-DNA alkyltransferases from Escherichia coli and rat liver to remove such alkyl groups from the O6-position of guanine. It was found that all of these adducts were removed by the alkyltransferases, but the branched alkyl chain iso-butyl- and iso-propyl adducts were removed very slowly. Also, when tested with a DNA substrate containing both O6-n-propylguanine and O6-iso-propylguanine, the alkyltransferases removed almost all of the n-propyl-adduct before the iso-propyl-adduct was attacked. Both alkyltransferases showed a decreasing rate of reaction as the size of the alkyl group increased, but there was a significant difference between the rat liver and E. coli alkyltransferase in the relative rates. The rat liver alkyltransferase repaired O6-methylguanine more slowly than the E. coli protein, but was considerably more rapid than the bacterial equivalent when acting on n-propyl- and n-butyl-adducts. The relative rates of repair were methyl much greater than ethyl greater than n-propyl greater than n-butyl greater than iso-propyl, iso-butyl for the E. coli alkyl-transferase and methyl greater than ethyl, n-propyl greater than n-butyl greater than iso-propyl, iso-butyl greater than 2-hydroxyethyl for the rat liver protein. These results indicate that differential rates of repair may contribute to the relative risks of carcinogenesis and mutagenesis by exposure to alkylating agents of different size and that rates of repair may be species specific and must be determined from specific measurements rather than extrapolated from data on other organisms.

Partially purified preparations of O6-alkylguanine-DNA alkyltransferase from rat liver and E. coli were tested for their ability to repair O4-methylthymine in a methylated poly(dT) X poly(dA) substrate. The bacterial preparation readily carried out this reaction, but no loss of O4-methylthymine was obtained with the rat liver protein. These results indicate a significant difference in specificity between the mammalian and bacterial proteins which could have important consequences for carcinogenesis and mutagenesis by alkylating agents in mammalian cells.

The rates of loss of O6-methylguanine and O6-ethylguanine from rat liver DNA were determined over a time period of 15 min to 4 hr after various doses (5 micrograms/kg to 2 mg/kg) of dimethylnitrosamine and diethylnitrosamine which produced total amounts of these adducts in the range of 300 to 16,000 molecules/cell. This amount is considerably less than the content of O6-alkylguanine-DNA alkyltransferase protein (approximately 60,000 molecules/hepatocyte), and during the time period studied, the adducts were found to be lost with pseudo-first order kinetics. The half-life for O6-methylguanine was 47 min. O6-Ethylguanine was removed 3.6 times more slowly with a half-life of 172 min. The ability of partially purified rat liver O6-alkylguanine-DNA alkyltransferase to remove O6-methylguanine and O6-ethylguanine from [3H]alkyl-labeled DNA substrates in vitro was measured, and it was found that O6-methylguanine was removed 3.4 times more rapidly than was O6-ethylguanine. These results are consistent with the hypothesis that most, if not all, of the repair of these adducts which occurs within the first 4 hr after treatment is due to the alkyltransferase protein. Diethylnitrosamine, which is slightly more potent as a carcinogen to rat liver, produced a total amount of O6-ethylguanine of 3.7 mumol/mol guanine/mg compared to O6-methylguanine (28 mumol/mol guanine/mg) given by dimethylnitrosamine. The slower rate of loss of the ethyl adduct is not sufficient to account for this difference, and the results, therefore, support the concept that other DNA adducts (possibly O-alkylpyrimidines) contribute to the initiation of tumors by diethylnitrosamine. Preliminary evidence that the rat liver alkyltransferase can also remove hydroxyethyl groups from DNA at a rate slower than removal of ethyl groups was also obtained. Bacterial O6-alkylguanine-DNA alkyltransferase was shown to remove methyl, ethyl, and hydroxyethyl groups from the O6 position of guanine in DNA using fluorescence detection to quantitate these adducts. The bacterial protein removed methyl groups very rapidly but was much slower than the rat liver protein on the larger adducts. These results suggest that the relative rates of repair of different alkyl groups may be species specific and must be determined experimentally in the cell of interest before conclusions concerning biological effects can be drawn.

We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the genome. Chinese hamster ovary cells were exposed to dimethyl sulfate, and the isolated DNA was treated with an appropriate restriction endonuclease. The DNA was heated to convert remaining N-methylpurines to apurinic sites to render them alkaline-labile. Duplicate samples heated in the presence of methoxyamine to protect the apurinic sites from alkaline hydrolysis provided controls to assess total DNA. After alkaline hydrolysis, agarose gel electrophoresis, Southern transfer, and probing for the fragment of interest, the ratios of band intensities of the test DNA sample to its methoxyamine-treated control counterpart were calculated to yield the percentage of fragments containing no alkaline-labile sites. The frequency of N-methylpurines was measured at different times after dimethyl sulfate treatment to study repair. We found no differences between the rates of repair of N-methylpurines in the active dihydrofolate reductase gene and a nontranscribed region located downstream from it in treated cells. Also, similar rates of repair were observed in the transcribed and nontranscribed strands of the gene, in contrast to previous results for the removal of cyclobutane pyrimidine dimers. Thus, there does not appear to be a coupling of N-methylpurine repair to transcription in Chinese hamster ovary cells. However, the repair in the dihydrofolate reductase domain appears to be somewhat more efficient than that in the genome overall. Our method permits the quantifying at the defined gene level of abasic sites or of any DNA adduct that can be converted to them.