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, 95 (9), 5172-7

Homology-directed Repair Is a Major Double-Strand Break Repair Pathway in Mammalian Cells

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Homology-directed Repair Is a Major Double-Strand Break Repair Pathway in Mammalian Cells

F Liang et al. Proc Natl Acad Sci U S A.

Abstract

Mammalian cells have been presumed to repair potentially lethal chromosomal double-strand breaks (DSBs) in large part by processes that do not require homology to the break site. This contrasts with Saccharomyces cerevisiae where the major DSB repair pathway is homologous recombination. Recently, it has been determined that DSBs in genomic DNA in mammalian cells can stimulate homologous recombination as much as 3 or 4 orders of magnitude, suggesting that homology-directed repair may play an important role in the repair of chromosomal breaks. To determine whether mammalian cells use recombinational repair at a significant level, we have analyzed the spectrum of repair events at a defined chromosomal break by using direct physical analysis of repair products. When an endonuclease-generated DSB is introduced into one of two direct repeats, homologous repair is found to account for 30-50% of observed repair events. Both noncrossover and deletional homologous repair products are detected, at approximately a 1:3 ratio. These results demonstrate the importance of homologous recombination in the repair of DSBs in mammalian cells. In the remaining observed repair events, DSBs are repaired by nonhomologous processes. The nonhomologous repair events generally result in small deletions or insertions at the break site, although a small fraction of events result in larger chromosomal rearrangements. Interestingly, in two insertions, GT repeats were integrated at one of the broken chromosome ends, suggesting that DSB repair can contribute to the spread of microsatellite sequences in mammalian genomes.

Figures

Figure 1
Figure 1
DSB repair substrate DRneo. (A) The DRneo substrate contains the 18-bp cleavage site for the rare-cutting endonuclease I-SceI (black bar) inserted in the S2neo gene. The homology between the S2neo gene and the 685-bp 3′ neo gene is indicated by the stippling. (B) Two outcomes of DSB-induced homologous recombination that produce a neo+ gene.
Figure 2
Figure 2
Southern blot analysis of pooled DSB-induced recombinants. Genomic DNA from the parental CHO-K1 DRA10 cell line and a pool of neo+ recombinants derived from expression of I-SceI was cleaved with either XhoI–NcoI (X/N) or XhoI–HindIII (X/H). The 5′ neo gene probe (probe A, see Fig. 1) has the same amount of homology to both types of recombination products.
Figure 3
Figure 3
Physical analysis of DSB repair events. (A) Scheme for PCR analysis. Genomic DNA was prepared from pools of cells that were transfected with the I-SceI expression vector and incubated in nonselective media for various times. The neo gene was subsequently amplified with primers 1 and 2, and the amplified product was cleaved with NcoI and I-SceI to detect homologous (NcoI+/I-SceI) and nonhomologous (NcoI/I-SceI) repair products. Nonhomologous repair could result in deletions (Δ) or insertions (++). (B) PCR analysis of genomic DNA that was not cut in vitro by I-SceI (Upper) or was cut in vitro with I-SceI (Lower) before PCR amplification. After PCR amplification, DNA was electrophoresed either uncleaved or cleaved with the indicated endonucleases and then probed with the entire neo gene probe (see Fig. 1, probe B). Genomic DNA preparations were 0, 4, or 24 h after transfection, as indicated.
Figure 4
Figure 4
Southern blot analysis of randomly isolated clones. Genomic DNA from cells that were electroporated with pCMV3xnlsI-SceI (A) or pCMVlacZ (B) and cloned in nonselective media was cleaved with XhoI–I-SceI–HindIII to detect popout (PO), gene conversion (GC), and nonhomologous end-joining (EJ) products. The variation in intensities of the hybridization signal in some lanes is caused by unevenness in the amount of genomic DNA loaded. The entire neo gene was used as probe. In clone 33, an undetermined type of repair event occurred, indicated by a ?.
Figure 5
Figure 5
Southern blot analysis of subclones from a clone containing a mixed genotype. Genomic DNA was cleaved with XhoI–I-SceI–HindIII and probed with the entire neo gene.
Figure 6
Figure 6
Sequence analysis of nonhomologous repair products. (A) Sequences of nonhomologous rejoining products derived from the S2neo gene. The underlined nucleotides in deletion products 5 and 7.2 indicate sequence overlap between the two ends at the junction. Bases from the ATAA I-SceI overhang are duplicated in insertions 11, 10, and 16X (indicated in lowercase letters at the break site). Note that some clones have two different repair products (i.e., clone 7, deletion 7.2 and insertion 7.1; clone 16, insertion 16X and a PO event not shown; and clone 35, deletion 35.1 and a PO event not shown). (B) Diagram of insertion products. Insertions are indicated by the stippled bars except for the insertions of the GT repeats (lined bars) and the simian virus 40 (SV40) origin region (arrows in 16X). The SV40 sequences are derived from three nearby regions that were joined together at a 6-bp sequence overlap and a 13-bp palindromic repeat, as shown.
Figure 7
Figure 7
Summary of DSB repair events derived from random clone analysis. Number of each type of repair event is indicated. PO, popout recombination; GC, noncrossover gene conversion; Δ, deletional rejoining; ++, insertional rejoining; Gr, gross chromosomal rearrangement.

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