The inhibition of repair in UV irradiated human cells

Abstract

We have used three different assay procedures to determine the effects of hydroxyurea on excision repair in UV-irradiated HeLa cells. The results were as follows. (a) At the cytological level, incubation of UV-irradiated metaphase cells with hydroxyurea caused chromosome decondensation. (b) Using a modified alkaline sucrose gradient sedimentation technique involving minimal lysis before centrifugation, we found a marked retardation in the sedimentation of DNA from UV-irradiated cells incubated for a short period with hydroxyurea. (c) The effect of hydroxyurea on the incorporation of[3H]thymidine by UV-irradiated G1 cells was found to depend on the concentration of thymidine present in the medium. Normal primary human cells resemble HeLa cells in the response of chromosomes and DNA to UV plus hydroxyurea. Xeroderma pigmentosum cells, deficient in excision repair, are not sensitive to hydroxyurea in our assays. Chromosome decondensation and retarded DNA sedimentation occur also after incubation of irradiated HeLa cells with deoxyadenosine, but not thymidine, at concentrations which inhibit semiconservative DNA synthesis. The effects of hydroxyurea or deoxyadenosine on chromosomes and DNA are not seen if all four deoxyribonucleoside precursors of DNA are supplied exogenously. These results point to an inhibition of repair DNA synthesis by hydroxyurea (or deoxyadenosine), at the level of the supply of DNA precursors, i.e. in the same way that these agents inhibit semiconservative DNA synthesis. In the presence of these inhibitors, single-strand gaps accumulate in the DNA.