Bacteriophage T7 RNA polymerase transcription elongation is inhibited by site-specific, stereospecific benzo[c]phenanthrene diol epoxide DNA lesions.

Benzo[c]phenanthrene diol epoxide (B[c]PhDE), the ultimate carcinogenic metabolite of the environmental pollutant benzo[c]phenanthrene, reacts with DNA primarily at the exocyclic amino groups of purines, forming B[c]PhDE-DNA adducts that differ in their stereochemical configurations and their effect on biological processes such as transcription. To determine the effect of these stereoisomers on RNA synthesis, in vitro T7 RNA polymerase transcription assays were performed using DNA templates modified on the transcribed strand by either a site-specific (+)-trans- or (-)-trans-anti-B[c]PhDE-N(6)-dA lesion located within the sequence 5'-CTCTCACTTCC-3'. The results show that both (-)-trans-anti-B[c]PhDE-N(6)-dA and (+)-trans-anti-B[c]PhDE-N(6)-dA block RNA synthesis. Furthermore, both B[c]PhDE-dA stereoisomeric adducts lead to lower levels of initiation of transcription relative to that observed using an unmodified DNA template. In contrast to these results, placement of the adduct on the nontranscribed strand within the template does not impede transcription elongation. In addition to the assessment of the effect of the lesions on transcription elongation, the resulting transcripts were characterized in terms of their base composition. A high level of base misincorporation is detected at the 3'-ends of truncated transcripts, with guanosine being most frequently incorporated opposite the modified nucleotide rather than the expected uridine. This result supports the notion that translocation past a modified base in a DNA template relies in part on correct base incorporation, and suggests that stalling of RNA polymerases at damaged sites in DNA may well be dependent on both the presence of the lesion and the base which is incorporated opposite the modified nucleotide.

Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

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.

Transcription and DNA damage: a link to a kink.

Living organisms are constantly exposed to a variety of naturally occurring and man-made chemical and physical agents that pose threats to health by causing cancer and other illnesses, as well as cell death. One mechanism by which these moieties can exert their toxic effects is by inducing modifications to the genome. Such changes in DNA often result in the formation of nucleotides not normally found in the double helix, bases containing covalent chemical alterations, single- and double-strand breaks, and interstrand and intrastrand cross-links. When these lesions are present during replication, mutations often result in the newly synthesized DNA. Likewise, when such damage occurs in a gene, transcription elongation, and hence expression, can be adversely affected because of pausing or arresting of the RNA polymerase at or near the altered site; this could result in the synthesis of a defective RNA molecule. It has become increasingly clear that transcription and DNA damage are intimately linked, since the removal of certain adducts from the genome is highly dependent on their location. When such lesions are present on the transcribed strand of actively expressed genetic loci, they are better cleared from that strand when compared to the complementary DNA or other quiescent regions. This process is called transcription-coupled DNA repair, and it modulates the mutagenic spectrum of many DNA-damaging agents. Furthermore, based upon evidence from systems in which it is absent, this process has a profound effect on ameliorating the adverse consequences of exposure to many environmentally relevant genotoxins. The precise cellular pathway that mediates the preferential clearance of DNA damage from active genetic loci has not yet been established, but it appears to be effected by a repertoire of proteins that are also involved in other DNA repair pathways and transcription as well as some factors that might be unique to it. Because a cellular process as indispensable as gene expression can be thwarted by the presence of DNA damage, an understanding of the mechanism underlying transcription-coupled DNA repair is relevant to the continued discernment of how environmental genotoxins endanger human health.

Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo[a]pyrenediol epoxide-modified DNA.

DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur. Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome. The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest. The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase. We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine. However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

3-Methyladenine and 7-methylguanine exhibit no preferential removal from the transcribed strand of the dihydrofolate reductase gene in Chinese hamster ovary B11 cells.

The removal of cylclobutane pyrimidine dimers from cellular DNA occurs preferentially in actively transcribed genes of cells subjected to ultraviolet radiation. In contrast, reports concerning the transcription-dependent repair of N-methylpurines formed in cellular DNA following exposure to methylating agents are quite conflicting, with some studies suggesting that no biased clearance of these lesions occurs and others indicating that preferential removal of these adducts transpires in active genetic loci. Even in the cases where no preferential clearance was demonstrated, a slight but statistically insignificant biased removal of N-methylpurines from the transcribed strand of active genes was often evident. We proposed that these results might be due to the preferential clearance of only one of the two principal N-methylpurines formed, 3-methyladenine, or to the source of the methylating species to which the cells were exposed. Therefore, we investigated the clearance of 3-methyladenine and 7-methylguanine as individual lesions from the amplified dihydrofolate reductase gene of Chinese hamster ovary cells, and we examined the gene-specific removal of N-methylpurines formed by several different methylating agents as well. We observed no biased clearance of 3-methyladenine toward the transcribed strand of the locus being examined. This result indicates that any minor gene-specific preferential repair that has been observed previously for N-methylpurines in toto--which actually reflects the removal of the predominant methylated purine 7-methylguanine--is not due to biased clearance of the transcription-inhibiting 3-methyladenine lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-specific benzo[a]pyrene diol epoxide-DNA adducts inhibit transcription elongation by bacteriophage T7 RNA polymerase.

Benzo[a]pyrene, an extremely potent procarcinogen and mutagen, is metabolized to a variety of products, including the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene. This product of biotransformation reacts with DNA, forming a series of adducts principally at the N2 position of guanine that differ in their stereochemistry and exhibit unique biological properties. In order to gain a better understanding of the effects on RNA synthesis of these adducts, we used purified bacteriophage T7 RNA polymerase to transcribe a series of templates containing one of four stereoisomerically pure BPDE-guanine lesions--(+)-trans-,(-)-trans-,(+)-cis-anti-N2-BPDE-guanine--or no damaged bases. To construct suitable double-stranded oligodeoxynucleotides for these studies, we annealed an 11-mer containing a site-specific stereoisomerically pure N2-BPDE-guanine adduct, a 37-mer, and a 10-mer to a complementary 58-base sequence of single-stranded DNA. The oligomers were ligated, purified, and reannealed. The resulting DNA template contained the promoter for T7 RNA polymerase and a BPDE adduct at position +16 following the transcription initiation site. The results of the transcription assays clearly demonstrate that each of the adducts inhibits elongation by T7 RNA polymerase, but they do so to significantly different extents, depending on the stereochemical characteristics of the BPDE-modified guanine. The order of inhibition is (+)-trans > (-)-trans > (+)-cis > (-)-cis, when the amount of full-length transcript for each is compared to that obtained for an unmodified template. Furthermore, premature termination of RNA synthesis occurs at or near the site of the BPDE lesion as evidenced by the formation of discrete, truncated transcripts. These results might be related to the fact that the pyrenyl moiety of the trans-BPDE adducts is situated in the minor groove of double-stranded DNA, but is quasi-intercalated into the double helix in the case of the cis stereoisomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Intragenomic repair heterogeneity of DNA damage.

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.

Two expressed human genes sustain slightly more DNA damage after alkylating agent treatment than an inactive gene.

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.

Lack of sequence-specific removal of N-methylpurines from cellular DNA.

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.

Repair of N-methylpurines in specific DNA sequences in Chinese hamster ovary cells: absence of strand specificity in the dihydrofolate reductase gene.

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.

Inhibition of O6-alkylguanine-DNA-alkyltransferase by metals.

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.