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.     

Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

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.     

Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli

An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli. The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI (Schaaper, R.M., Dunn, R.L., and Glickman, B.W. (1987) J. Mol. Biol. 198, 187-202). On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed. A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand. This bias is reduced in an excision repair deficient (UvrB-) strain. In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand. This bias is also markedly reduced in the UvrB- strain. In light of recent work Mellon and Hanawalt (Mellon, I., and Hanawalt, P.C. (1989) Nature 342, 95-98) describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E. coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis.     

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.