The Use of Recombinant DNA Techniques to Study Radiation-induced Damage, Repair and Genetic Change in Mammalian Cells

Summary

A brief Introduction is given to appropriate elements of recombinant DNA techniques and applications to problems in radiobiology are reviewed with illustrative detail. Examples are included of studies with both 254 nm ultraviolet light (u.v.) and ionizing radiation (i.r.) and the review progresses from the molecular analysis of DNA damage in vitro through to the nature of consequent cellular responses. The section on the Molecular distribution of DNA damage (§ 2) focuses on the use of defined DNA molecules to assess the nature, sites and frequency of radiation damage. Recombinant DNA techniques have also been used in the study of enzyme—DNA interactions, to comment upon the rôle of specific types and sites of damage in producing cellular responses. The use of DNA-mediated gene transfer to assess damage and repair (§ 3) indicates that recombinant DNA molecules can be used to implicate (or reject) specific types of DNA damage in gene inactivation. Some gene-transfer assays may also be able to confirm the presence of specific repair functions in mammalian cells. Restriction endonucleases are essential for the construction of recombinant DNA molecules, but their ability to cut DNA at specific sequences is also being exploited to implicate the double-strand break as an important type of damage leading to the well-characterized responses of irradiated cells. The DNA double strand break: use of restriction endonucleases to model radiation damage (§ 4) documents experiments showing that blunt-ended cuts introduced into cellular DNA are able to produce chromosome aberrations and cell death. Assays based upon the introduction of restriction endonuclease-cut plasmids into radiosensitive and normal cells suggest that sensitivity is in some instances, e.g. the radiosensitive disorder ataxia-telangiectasia, a result of excessive degradation of DNA around broken ends. Identification and cloning of DNA repair genes (§ 5) reviews the successful cloning of one human repair gene and the putative identification of others, as well as the lack of success in identifying genes complementing radiosensitive human disorders. Analysis of radiation-induced genetic change (§ 6) links the types of DNA damage observed in defined DNA molecules with the types of mutations occurring in irradiated prokaryotes. In mammalian cells recombinant DNA techniques have allowed the nature of mutational changes to be determined for the first time: to date it seems that u.v. produces mainly small (point) mutations while i.r. produces mainly large changes (deletions/rearrangements). A final section comments on the usefulness of recombinant DNA techniques for present and future studies in radiobiology.