The influence of DNA double-strand break structure on end-joining in humancells

DNA end-joining is the major repair pathway for double-strand breaks (DSBs) in higher eukaryotes. To understand how DSB structure affects the end-join ing process in human cells, we have examined the in vivo repair of lineariz ed plasmids containing complementary as well as several different configura tions of non-complementary DNA ends. Our results demonstrate that, while co mplementary and blunt termini display comparable levels of error-free rejoi ning, end-joining fidelity is decreased to varying extents among mismatched non-complementary ends. End structure also influences the kinetics of repa ir, accurately recircularized substrates for blunt and complementary termin i being detected significantly earlier than for mismatched non-complementar y ends. These results suggest that the end-joining process is composed of a n early component, capable of efficiently repairing substrates requiring a single ligation event, and a late component, involved in the rejoining of c omplex substrates requiring multiple processing steps. Finally, these two t ypes of repair events may have different genetic requirements as suggested by the finding that exposure of cells to wortmannin, a potent inhibitor of phosphatidylinositol 3-related kinases (PI 3-related kinases), blocks the r epair of complex substrates while having little or no effect on those requi ring a simple ligation event.