Recombination of parent and daughter strand DNA after UV-irradiation in mammalian cells

Abstract

The mechanism by which mammalian cells replicate DNA containing pyrimidine dimers is poorly understood. When DNA synthesis is initiated after UV-irradiation in bacteria, parental DNA containing pyrimidine dimers has been shown to ‘exchange’ into the daughter strand DNA by a recA-dependent mechanism^1–3. In earlier experiments, when growing mammalian cells were UV-irradiated and then incubated with labelled thymidine, pyrimidine dimers were initially interpreted to be in the newly synthesized DNA⁴, but later were found to be only adjacent to newly synthesized DNA in daughter strand present before UV treatment⁵. A way to avoid these problems of interpretation would be to use cells in G₀ or G₁ which are not synthesizing DNA at the time of irradiation. UV damage could then be detected in a very sensitive quantitative assay such as that recently described using alkaline elution and an endonuclease preparation from Micrococcus luteus⁶. I have now used this approach and report that up to 3 dimers per 10⁹ daltons of daughter strand DNA could be detected 34–45 h after UV-irradiation in human peripheral blood lymphocytes (PBL), normal human fibroblasts, group A xeroderma pigmentosum (XP) fibroblasts and mouse 3T3 cells. This represents approximately 1–3% of the dimers present in the parent strand at this time after UV.