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

Abstract

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.

S-phase progression stimulates both the mutagenic KU-independent pathway and mutagenic processing of KU-dependent intermediates, for nonhomologous end joining

“…The present data lend molecular support to these observations by directly measuring both efficiency and accuracy of NHEJ itself, in a chromosome context. These data are also in line with the notion that in human lymphoblastoid cells, NHEJ repair of I-SceI-induced DSB mainly results in small or no deletions (Honma et al, 2003(Honma et al, , 2007. In addition our data also revealed that the accuracy of end joining is affected by the dynamic progression through S phase.…”

Chromosomal aberrations induced by double strand DNA breaks

“…Nonconservative repair processes abrogated the activity of the reporter genes and enabled cells to survive in selection media. In another experimental system [19], a DSB was introduced in an intron of the endogenous Tk gene of a human lymphoblastoid cell line, and aberrant DNA repair events were recovered by selecting for cells that had lost Tk activity. However, this assay favors the recovery of large scale changes, such as long deletions, and does not provide a means of assessing the frequency of short deletions.…”

LOH Analyses for Biological Effects of Space Radiation: Human Cell Culture in "Kibo" of International Space Station

“…Since genetic changes at chromosome level are considered to be caused by chromosome break or DNA double-strand break (DSB), the repair of DSB was evaluated by our model system (Fig. 6) (Honma et al, 2003). The repair of DSBs is considered to be mainly due to two pathways, non homologous end-joining (NHEJ) and homologous recombination (HR).…”

“…The repair of DSBs is considered to be mainly due to two pathways, non homologous end-joining (NHEJ) and homologous recombination (HR). The details of methodologies for evaluating the above DSB repair were already described in our previous paper (Honma et al, 2003, Honma etal., 2007, and Yatagai et al sequence containing the recognition sequence for the restriction enzyme I-SceI was introduced at the region upstream of exon 5 in the TK + allele of TK6 cells. The cells containing this insert were designated TSCE5.…”