Repair of individual DNA strands in the hamster dihydrofolate reductase gene after treatment with ultraviolet light, alkylating agents, and cisplatin.

We have analyzed gene-specific and strand-specific DNA damage and repair in the dihydrofolate reductase gene in hamster cells. Cells were UV-irradiated or treated with two types of chemotherapeutics, alkylating agents or cisplatin. UV-induced pyrimidine dimers were detected using a previously published technique in which the T4 endonuclease V enzyme is used to create nicks at the lesion sites. 6-4 photoproducts were detected in a similar assay using ABC excinuclease after prior reversal of the pyrimidine dimers with photolyase. Adducts formed by the alkylating agents nitrogen mustard and dimethyl sulfate were quantitated by generating strand breaks at basic sites after neutral depurination. Cisplatin-induced intrastrand adducts were detected with ABC excinuclease, and cisplatin interstrand cross-links were detected using a denaturation-reannealing reaction before electrophoresis. In accord with previous reports by other investigators, we find distinct strand specificity of the repair of pyrimidine dimers after UV; the transcribed strand was much more efficiently repaired than the nontranscribed strand. In contrast, there was little or no strand bias in the repair of the 6-4 photoproducts. For alkylating agents, a slight bias toward repair in the transcribed strand was found after treatment with nitrogen mustard, but there appeared to be no bias in the repair after treatment with dimethyl sulfate. Cisplatin interstrand cross-links are repaired with equal efficiency from the two strands, but the more common cisplatin-induced lesion, the intrastrand adduct, is preferentially repaired from the transcribed strand. In conclusion, there is strand bias in the repair of pyrimidine dimers and cisplatin intrastrand adducts, but the strand specificity of repair may not be a general feature for all DNA lesions, as we found little or no strand bias in the repair of other lesions studied.

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells.

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.

Human repair gene restores normal pattern of preferential DNA repair in repair defective CHO cells.

The pattern of preferential DNA repair of UV-induced pyrimidine dimers was studied in repair-deficient Chinese hamster ovary (CHO) cells transfected with the human excision repair gene, ERCC-1. Repair efficiency was measured in the active dihydrofolate reductase (DHFR) gene and in its flanking, non-transcribed sequences in three cell lines: Wild type CHO cells, a UV-sensitive excision deficient CHO mutant, and the transfected line of the mutant carrying the expressed ERCC-1 gene. The CHO cells transformed with the human ERCC-1 gene repaired the active DHFR gene much more efficiently than the non-transcribed sequences, a pattern similar to that seen in wild type CHO cells. This pattern differs from that previously reported in CHO cells transfected with the denV gene of bacteriophage T4, in which both active and non-transcribed DNA sequences were efficiently repaired (Bohr and Hanawalt, Carcinogenesis 8: 1333-1336, 1987). The ERCC-1 gene product may specifically substitute for the repair enzyme present in normal hamster cells while the denV product, T4 endonuclease V, does not be appear to be constrained in its access to inactive chromatin.

DNA repair in the metallothionein gene increases with transcriptional activation.

We have studied DNA repair in the Chinese Hamster Ovary (CHO) metallothionein (MT) gene after UV-light induced damage. The repair was examined comparatively with or without transcriptional activation of the gene by incubation in the presence of the heavy metal ZnCl2. Whereas the repair efficiency was very low in the uninduced state, it increased significantly after induction of the gene. The presence of ZnCl2 did not appear to change other repair parameters in the cells. The overall genome DNA repair efficiency after UV irradiation was similar whether or not the gene was induced and the preferential DNA repair pattern in the essential dihydrofolate reductase (DHFR) gene which we have previously described was unaffected by the presence of ZnCl2. Based upon repair analysis in two different restriction fragments containing the MT I gene, we conclude that the region of efficient repair after induction is considerably larger than the 1 kb size of the gene. The results suggest that the accessibility of a genomic region to DNA repair enzymes may be regulated by the local chromatin structure in a dynamic manner.

Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells.

The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin.

Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene.

The survival of UV-irradiated mammalian cells is not necessarily correlated with their overall capacity to carry out DNA repair. Human cells typically remove 80% of the pyrimidine dimers produced by a UV dose of 5 J/m2 within 24 hr. In contrast, a Chinese hamster ovary (CHO) cell line survives UV irradiation equally well while removing only 15% of the dimers. Using a newly developed technique to measure dimer frequencies in single-copy specific sequences, we find that the CHO cells remove 70% of the dimers from the essential dihydrofolate reductase (DHFR) gene but only 20% from sequences located 30 kilobases or more upstream from the 5' end of the gene in a 24-hr period. Repair-deficient human cells from xeroderma pigmentosum complementation group C (XPC) are similar to the CHO cells in overall repair levels, but they are extremely sensitive to killing by UV irradiation. In the XPC cells, we find little or no repair in the DHFR gene; in contrast, in normal human fibroblasts and epidermal keratinocytes, greater than 80% of the dimers induced in the gene by 20 J/m2 are removed in 24 hr. Since the CHO and normal human cells exhibit similar UV resistance, much higher than that of XPC cells, our findings suggest a correlation between efficient repair of essential genes and resistance to DNA-damaging agents such as UV light.

DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall.

DNA repair was measured in the dihydrofolate reductase gene in Chinese hamster ovary cells, amplified for the gene, by quantitating pyrimidine dimers with a specific UV-endonuclease. More than two thirds of the dimers had been removed from a 14.1 kb restriction fragment of the gene by 26 hr after irradiation (20 J/m2), while little removal was detected in fragments upstream of the gene and only 15% were removed from the genome overall. This suggests that damage processing can vary according to function or activity of affected sequences, which has general implications for correlations of DNA repair with survival and mutagenesis. Perhaps preferential repair of vital sequences facilitates UV-resistance of these cells despite low overall repair levels.

Nature of DNA repair synthesis resistant to inhibitors of polymerase alpha in human cells.

Arabinocytidine and aphidicolin are inhibitors of alpha-DNA polymerase that have been shown to affect both normal DNA replication and repair synthesis in mammalian cells. In contradiction to the prevalent hypothesis that these inhibitors merely slow the polymerization rate at incision sites near lesions, our results suggest that the repair synthesis resistant to inhibitors is mediated by a separate pathway. Repair synthesis in contact-inhibited human cells following UV irradiation was inhibited 75-80% by arabinocytidine or aphidicolin, and most of the repair patches were not ligated into parental DNA, as judged by an enzymatic assay. However, the patches were not demonstrably shorter than those in untreated cells. Even following low-UV doses at which no inhibition of repair synthesis by the inhibitors was observed, a majority of the patches were not ligated. DNA polymerase beta is implicated in this alternate pathway, both by the known specificity of the inhibitors and by evidence from their sensitivity to S1 nuclease that the patches arise from displacement synthesis. The unligated patches are not degraded in vivo and eventually become ligated into parental DNA, very slowly in the presence of inhibitors but much more rapidly following their removal. Thus, under conditions of alpha-polymerase inhibition, a limited number of normal length repair patches are made, apparently by displacement synthesis, leaving displaced strands that remain substantially undegraded.