Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine on the kinetics of repair
Erixon, K.; Ahnström, G.
Mutation Research 59(2): 257-271
1979
ISSN/ISBN: 0027-5107 PMID: 35744 Document Number: 140712
A simple and sensitive technique for detection of strand breaks in DNA was further developed. The method was used to follow UV-induced excision-repair in human fibroblasts. It was possible to study the kinetics of enzymic reactions in intact cells, in which strand breaks in DNA are produced and sealed again. Hydroxyurea, 5-fluorodeoxyuridine and 1-.beta.-D-arabinofuranosylcytosine [ara C], potent inhibitors of DNA synthesis, drastically increased the number of breaks observed during the repair process. This was probably due to a decreased polymerase activity, which will cause the strand breaks formed by endonuclease to remain open longer. The initial rate of strand-break formation did not seem to be influenced by hydroxyurea or araC, and was about 4000 breaks/min in a diploid genome, at a dose of 20 J/m2. After 5-30 min, depending on the dose of UV, the number of breaks reached a maximum and started to decrease again. Hydroxyurea decreased the rate of polymerization in the sites under repair. There was no concomitant reduction of repair-induced incorporation of [3H]thymidine and no reduction of the excision of pyrimidine dimers. The action of the polymerase was probably not a rate-limiting event, but rather an earlier step. The endonucleolytic activity possibly determined the rate of repair. The endonuclease and polymerase cannot be bound in a permanent complex. Under certain assumptions, the time for repair of a site, i.e., the time from incision to final ligase sealing, is estimated as 3-10 min. Essentially no breaks were produced in xeroderma pigmentosum cells belonging to complementation group A, and there was no enhancement by hydroxyurea. Cells from the variant type of xeroderma pigmentosum behaved like normal cells in this respect.