Comparison of repair of DNA double-strand breaks in identical sequences inprimary human fibroblast and immortal hamster-human hybrid cells harboringa single copy of human chromosome 11

We have optimized a pulsed-field gel electrophoresis assay that measures in duction and repair of double-strand breaks (DSBs) in specific regions of th e genome (Lobrich et al,, Proc. Natl, Acad, Sci, USA 92, 12050-12054, 1995) , The increased sensitivity resulting from these improvements makes it poss ible to analyze the size distribution of broken DNA molecules immediately a fter the introduction of DSBs and after repair incubation. This analysis sh ows that the distribution of broken DNA pieces after exposure to sparsely i onizing radiation is consistent with the distribution expected from randoml y induced DSBs, It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misre joining events, since novel restriction fragments both larger and smaller t han the original fragment are generated after repair. This improved assay w as also used to study the kinetics of DSB rejoining and the extent of misre joining in identical DNA sequences in human GM38 cells and human-hamster hy brid A(L) cells containing a single human chromosome Il, Despite the numero us differences between these cells, which include species and tissue of ori gin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kine tics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(I) phase of the ce ll cycle, Furthermore, DSBs were removed from the single-copy human chromos ome in the hamster A(L) cells with similar kinetics and misrejoining freque ncy as at a locus on this hybrid's CHO chromosomes. (C) 2000 by Radiation R esearch Society.