Repair of radiation-induced DNA strand breaks does not occur preferentially in transcriptionally active DNA.

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.     

Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells.

Removal of ultraviolet light induced cyclobutane pyrimidine dimers (CPD) from active and inactive genes was analyzed in cells derived from patients suffering from the hereditary disease Cockayne's syndrome (CS) using strand specific probes. The results indicate that the defect in CS cells affects two levels of repair of lesions in active genes. Firstly, CS cells are deficient in selective repair of the transcribed strand of active genes. In these cells the rate and efficiency of repair of CPD are equal for the transcribed and the nontranscribed strand of the active ADA and DHFR genes. In normal cells on the other hand, the transcribed strand of these genes is repaired faster than the nontranscribed strand. However, the nontranscribed strand is still repaired more efficiently than the inactive 754 gene and the gene coding for coagulation factor IX. Secondly, the repair level of active genes in CS cells exceeds that of inactive loci but is slower than the nontranscribed strand of active genes in normal cells. Our results support the model that CS cells lack a factor which is involved in targeting repair enzymes specifically towards DNA damage located in (potentially) active DNA.     

Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT gene.

DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.     

3-Methyladenine-DNA glycosylase: a probe for determining alkylation damage and repair in human fibroblasts

3-Methyladenine-DNA glycosylase was partially purified from human lymphoblasts and used as an enzymatic probe to assay the amounts of 3-methyladenine in DNA from cultured human fibroblasts after treatment with dimethyl sulfate. Aside from this specific alkylation product, the total number of alkylated bases was estimated after depurination by heating. Both enzyme-induced and heat-induced apurinic sites were converted to strand breaks and estimated after alkaline sucrose-gradient sedimentation. The results indicate that 3-methyladenine in cultured human fibroblasts is rapidly excised, with a half-life of about 2 hours. The rest of the alkylated purines (mostly 7-methylguanine) are removed much more slowly, with a half-life of about 20 hours.     

(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.     

Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.

A UV-resistant revertant (XP129) of a xeroderma pigmentosum group A cell line has been reported to be totally deficient in repair of cyclobutane pyrimidine dimers (CPDs) but proficient in repair of 6-4 photoproducts. This finding has been interpreted to mean that CPDs play no role in cell killing by UV. We have analyzed the fine structure of repair of CPDs in the dihydrofolate reductase gene in the revertant. In this essential, active gene, we observe that repair of the transcribed strand is as efficient as that in normal, repair-proficient human cells, but repair of the nontranscribed strand is not. Within 4 h after UV at 7.5 J/m2, over 50% of the CPDs were removed, and by 8 h, 80% of the CPDs were removed. In contrast, there was essentially no removal from the nontranscribed strand even by 24 h. Our results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs from the transcribed strands of expressed genes is essential for UV resistance.     

Removal of minor methylation products 7-methyl adenine and 3-methylguanine from DNA ofescherichia coli treated with dimethyl sulphate

Persistence of methylpurines in DNA methylated in vitro and in vivo inEscherichia coli WP2 cells, by dimethyl sulphate (DMS) was studied, with particular reference to the minor products 7-methyladenine and 3-methyl-guanine, not previously investigated in this respect, but known to be removed from DNA in vitro by spontaneous hydrolysis at neutral pH.The half-life of 7-methyladenine in vivo was relatively short (2.6 ± 0.2 h) but not significantly shorter than in vitro at pH 7.2, 37°C. The half-life of 3-methylguanine was 3.6 ± 0.3 h in vivo, markedly shorter than in vitro, where its stability was somewhat greater than that of 7-methylguanine. Enzymatic excision of 3-methylguanine was therefore indicated to occur inE. coli.Previous findings that 7-methylguanine is probably not enzymatically excised from DNA in vivo, whereas 3-methyladenine is rapidly removed, were confirmed, and additional support for the concept of enzymatic removal of 3-methyladenine was obtained by showing extensive inhibition of its removal from cells treated with iodoacetamide prior to methylation.It is suggested that methylations of adenine or guanine in DNA at N-3 constitute blocks to template activity of DNA and stimulate a “repair” response of enzymatic removal of 3-methylpurines. Possible valence bond structures for 3-methylpurine residues in DNA are discussed, leading to the suggestion that ionized forms with positively charged amino groups may be the most effective blocks to template activity.