Deletion, rearrangement, and gene conversion; genetic consequences of chromosomal double-strand breaks in human cells.

Chromosomal double-strand breaks (DSBs) in mammalian cells are usually repaired through either of two pathways: end-joining (EJ) or homologous recombination (HR). To clarify the relative contribution of each pathway and the ensuing genetic changes, we developed a system to trace the fate of DSBs that occur in an endogenous single-copy human gene. Lymphoblastoid cell lines TSCE5 and TSCER2 are heterozygous (+/-) or compound heterozygous (-/-), respectively, for the thymidine kinase gene (TK), and we introduced an I-SceI endonuclease site into the gene. EJ for a DSB at the I-SceI site results in TK-deficient mutants in TSCE5 cells, while HR between the alleles produces TK-proficient revertants in TSCER2 cells. We found that almost all DSBs were repaired by EJ and that HR rarely contributes to the repair in this system. EJ contributed to the repair of DSBs 270 times more frequently than HR. Molecular analysis of the TK gene showed that EJ mainly causes small deletions limited to the TK gene. Seventy percent of the small deletion mutants analyzed showed 100- to 4,000-bp deletions with a 0- to 6-bp homology at the joint. Another 30%, however, were accompanied by complicated DNA rearrangements, presumably the result of sister-chromatid fusion. HR, on the other hand, always resulted in non-crossing-over gene conversion without any loss of genetic information. Thus, although HR is important to the maintenance of genomic stability in DNA containing DSBs, almost all chromosomal DSBs in human cells are repaired by EJ.