REPAIR AND MISREPAIR OF SITE-SPECIFIC DNA DOUBLE-STRAND BREAKS BY HUMAN CELL-EXTRACTS

The rejoining by human cell extracts of a double-strand break induced by endonuclease treatment at one of several sites within a small DNA molecule was studied. Rejoining was found at each of 8 sites tested, but the rejoin efficiency varied with the nature of the break (e.g., breaks with cohesive ends were rejoined more efficiently than blunt-ended breaks). Extracts from primary and immortalized cell lines, as well as those from individuals with ataxia telangiectasia (A-T), showed the same pattern of relative rejoin efficiencies. However, mis-rejoining varied with the cell extract used, and was particularly elevated with two immortalized A-T cell lines. Mixing experiments showed that the mis-rejoining property of extracts could act in a semi-dominant fashion, depending on the individual efficiencies of the component extracts. The mis-rejoin mechanism involved deletion at sites of short direct repeats at various distances from the initial break site. A model of deletion formation (the strand-exposure and repair model) is restated to explain the sequence repeat dependence found, and is compared to models of homologous DNA recombination.