Coupled homologous and nonhomologous repair of a double-strand break preserves genomic integrity in mammalian cells

Abstract

DNA double-strand breaks (DSBs) may be caused by normal metabolic processes or exogenous DNA damaging agents and can promote chromosomal rearrangements, including translocations, deletions, or chromosome loss. In mammalian cells, both homologous recombination and nonhomologous end joining (NHEJ) are important DSB repair pathways for the maintenance of genomic stability. Using a mouse embryonic stem cell system, we previously demonstrated that a DSB in one chromosome can be repaired by recombination with a homologous sequence on a heterologous chromosome, without any evidence of genome rearrangements (C. Richardson, M. E. Moynahan, and M. Jasin, Genes Dev., 12:3831-3842, 1998). To determine if genomic integrity would be compromised if homology were constrained, we have now examined interchromosomal recombination between truncated but overlapping gene sequences. Despite these constraints, recombinants were readily recovered when a DSB was introduced into one of the sequences. The overwhelming majority of recombinants showed no evidence of chromosomal rearrangements. Instead, events were initiated by homologous invasion of one chromosome end and completed by NHEJ to the other chromosome end, which remained highly preserved throughout the process. Thus, genomic integrity was maintained by a coupling of homologous and nonhomologous repair pathways. Interestingly, the recombination frequency, although not the structure of the recombinant repair products, was sensitive to the relative orientation of the gene sequences on the interacting chromosomes.

FIG. 1

Interchromosomal recombination substrates. The 5′neo allele (A) was constructed by targeting 5′neo and the hyg gene to the pim-1 locus on chr.17. The forward orientation (F allele) of the 5′neo sequence is present in the F5′/3′ cell lines, and the reverse orientation (R allele) is present in the R5′/3′ cell lines, as indicated by the arrow orientation. The 3′neo allele (B) was constructed by targeting 3′neo and the HPRT gene to the Rb locus on chr.14. Wild-type (top) and targeted (bottom) alleles are diagrammed for each locus, with confirmatory Southern analyses presented using HincII and PstI for the 5′neo and the 3′neo alleles, respectively, and probing with sequences as indicated. An I-SceI site disrupts the 5′neo gene at the NcoI site. Two independently derived clones were analyzed for both F5′/3′ and R5′/3′, as indicated (F5′/3′, F12 and G12; R5′/3′, B3 and C11). Black bars, pim-1 (A) or Rb (B) exons. HII, HincII; Pst, PstI; Nco, NcoI. (C) The truncated 5′neo and 3′neo sequences. Distances are indicated in bp. 5′neo contains a promoter, and 3′neo contains the neo gene stop codon and a polyadenylation signal. There is 468 bp of overlap between the 5′neo and 3′neo sequences.

FIG. 2

DSB repair products. Restriction maps of parental (A) and repaired neo⁺ alleles (B). NcoI digestion is diagnostic for homologous repair of the I-SceI-generated DSB in either 5′neo allele. BglII/SacII digestion is used to characterize the type of recombination event. BglII/SacII fragments of variable size (B) indicate gene conversion tracts incorporating variable lengths of chr.14 sequences (+chr.14) and NHEJ at variable positions of chr.17. BII, BglII; N, NcoI; SII, SacII. (C) Southern blot analysis of representative clones. Note that the 3′neo allele is unchanged from the parental allele in each of the recombinant clones. The probe was a 5′neo gene fragment. (D) Position of PCR primers derived from chr.17 sequences that flank the repair junctions.

FIG. 3

Sequence analysis of repair junctions from class II neo⁺ clones. PCR products from the neo⁺ allele of the Fneo⁺/3′ (A) and Rneo⁺/3′ cell lines (B) were cloned and sequenced. Extension on chr.14 indicates the length of newly synthesized DNA incorporated at one end of the chr.17 break site beyond the neo stop codon. Thus, the total extension includes an additional 229 bp to the number indicated. Deletion on chr.17 indicates the number of base pairs deleted from the nonconverted end of the I-SceI break site prior to NHEJ. The sequences at the junctions of chr.14 and chr.17 are separated by a hyphen when there is no sequence overlap, underlined if microhomology is present, or indicated by the base pairs that were inserted at the junction.

FIG. 4

Visualization of a rare translocation by FISH using whole chromosome mouse chr.14 fluorescein isothiocyanate (green) and chr.17 Cy3 (red) probes (left panel) and DAPI (right panel). (A) Representative class IV clone having two normal chrs.14 and chrs.17. (B) Reciprocal translocation clone (Rneo⁺/3′ clone 17) having two normal chrs.14, one normal untargeted chr.17, and two hybrid translocation chromosomes involving chr.17 and an unidentified chromosome (red/unstained), as indicated by the arrows.

FIG. 5

Model for partial tandem gene duplications. Events can be initiated by a DSB within a repetitive element (gray box) on one chromosome, followed by invasion of one end into the same element on the homologue or sister chromatid. Repair synthesis extends downstream to duplicate sequences, shown here as exons 2 and 3 (white bars). Completion of the repair event occurs by NHEJ of the newly synthesized strands at or near the end of the broken chromosome. The result is a partial tandem gene duplication on one chromosome or chromatid, while the other chromosome or chromatid remains unaltered.