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Repair of ribosomal RNA genes in hamster cells after UV irradiation, or treatment with cisplatin or alkylating agents
Laboratory of Molecular Pharmacology, Division of Cancer Treatment, Bldg 37, Room 5C-25, National Cancer Institute NIH, Bethesda, MD 20892, USA
1Cancer Research Institute, Slovak Academy of Sciences Bratislava, Slovakia
2To whom correspondence should be sent at present address: Laboratory of Molecular Genetics, National Institutes on Aging, NIH, 4940 Eastern Ave, Baltimore, MD 21224, USA
We have measured the DNA damage formation and repair in the ribosomal and the dihydrofolate reductase (DHFR) genes after treatment of hamster cells with different types of DNA damaging agents. In mammalian cells, the ribosomal DNA (rDNA) is transcribed by RNA polymerase I, whereas the DHFR is transcribed by RNA polymerase II. Cells were treated with agents that induce different types of lesions, and that are known to be repaired via different pathways. We used UV (254 nm) irradiation, treatment with cisplatin and treatment with the alkylating agents nitrogen mustard (HN2) and methyl methanesulphonate (MMS). UV induced pyrimidine dimers were detected with the enzyme T4 endonuclease V, which creates nicks at the dimer sites; the breaks are then resolved and identified by denaturing electrophoresis and Southern blot. Intrastrand adducts formed by the alkylating agents HN2 and MMS were quantitated by generating strand breaks at abasic sites after neutral depurination. Interstrand crosslinks (ICL) formed by HN2 and cisplatin were detected by a denaturation-reannealing reaction before neutral agarose gel-electrophoresis. We find that the repair of the pyrimidine dimers is significantly less efficient in the RNA polymerase I transcribed rDNA genes than in RNA polymerase II transcribed DHFR gene at 8 and 24 h after irradiation. ICL and intrastrand adducts induced by HN2 are also removed more slowly from the rDNA than from the DHFR gene. In contrast, MMS induced intrastrand adducts and cisplatin induced ICL are repaired equally efficiently in the RNA polymerase I and RNA polymerase II transcribed genes. We conclude that for some types of DNA damage, there is less repair in the ribosomal genes than in the DHFR; but for other DNA lesions there is no difference. The difference in repair efficiency between the rDNA and the DHFR genes may reflect the different RNA polymerases involved in their transcription. It may, however, alternatively, reflect the different nuclear localization of these genes.
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