Measurement of M. luteus endonuclease-sensitive lesions by alkaline elution

Abstract

The UV-endonuclease approach to detect DNA damage has been combined with the alkaline elution technique with a resultant marked increase in sensitivity compared to the conventional method using alkaline sedimentation. DNA from UV-irradiated cells was digested on an inert filter with an extract from Micrococcus luteus and then analyzed by alkaline elution. Endonuclease-sensitive sites (endo-sites) were measured after doses of 0.08–.07 Jm −2 of UV-radiation. An estiamte of endo-site production with UV radiation, 0.27 endo-sites/10 8 daltons of DNA/0.1 Jm −2, was similar to that usually seen at higher doses by others. With repair incubation, approx. 50% of the endo-sites were removed in 4 h by normal human fibroblasts after 0.2 or 0.4 Jm −2; no appreaciable repair was seen in xeroderma pigmentosum fibroblasts from complementation group A after 24 h of repair incubation. No photoreaction of UV damage due to 0.4 Jm −2 was detected in normal human fibroblasts. The endonuclease preparation also recognized DNA damage produced by ionizing radiation or an alkylating agent. Approx. 0.4 endo-sites/10 8 daltons of DNA were detected after a dose of 1 krad and 1 endo-site/10 8 daltons was observed after exposure of human cells to 2.5 μM MNNG for 1.3 h. The lesions detected after MNNG treatment by the endonuclease preparation decreased with post-treatment incubation—T1/2 8 h. The kinetics of removal of the endo-sites induced by MNNG were similar in normal cells and human cells of the mer − phenotype which had been shown to be more sensitive by cell killing to alkylating-agent damage. This should prove to be a useful approach to study DNA damage and repair since the entire assay can be done in several hours and a very low level of damage (1 endo-site/2 × 10 0 daltons of DNA) can be detected.