Isolation and partial characterization of mutagen‐sensitive and DNA repair mutants of chinese hamster fibroblasts

Abstract

Through a new approach, we have sought to isolate ultraviolet light (UV)‐sensitive and DNA repair mutant Chinese hamster fibroblasts. The procedure consisted of 1) mutation induction by 5‐bromodeoxyuridine (BrdU)‐blacklight and UV treatments; 2) incorporation of ³H‐thymidine in repair‐proficient cells at high temperature (38.5°C) following UV damage; 3) cold holding (4.0°C) of these cells to induce tritium killing; and 4) recovery and testing of repair‐deficient and UV‐sensitive cells which have survived and formed colonies at low temperature (34.0°C). In our initial attempt at this protocol, we isolated 72 surviving colonies from 2 × 10⁷ cells plated for selection. Of the 72 colonies. 20 demonstrated potential interest and four were selected for extensive study. One, identified as UV^s−7, is slightly more sensitive to UV, but not sensitive to X rays or N‐acetoxy‐2‐acetylaminofluorene (NAc‐AAF). The mutant exhibits a highly reduced level of unscheduled DNA synthesis (UDS), as compared to the parental line. Two additional lines, UV^s−40 and UV^s−44, are sensitive to UV, X ray, N‐methyl‐N‐nitro‐N‐nitrosoguanidine (MNNG), and NAc‐AAF, but exhibit normal UDS. A fourth line, UV^r−23, has enhanced UDS, is resistant to UV, but exhibits no difference in sensitivity to X ray or NAc‐AAF. These mutants are all stable, and should be useful for the study of mammalian DNA repair processes and mechanisms of mutagenesis.