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. 1999 Dec;19(12):8353-60.
doi: 10.1128/MCB.19.12.8353.

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

Y Lin  1 , T Lukacsovich, A S Waldman
Affiliations

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

Y Lin et al. Mol Cell Biol. 1999 Dec.

Abstract

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.

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Figures

FIG. 1
FIG. 1
DNA substrates for DSB repair. (A) Schematic of a generic substrate. For simplicity, the DNA construct is shown in linear form as if linearized at the unique ClaI site in the vector. Inserted between two BamHI (B) sites is a 2.5-kb DNA fragment containing a TK gene disrupted by a 22-bp oligonucleotide (stippled segment) containing the 18-bp recognition site for yeast endonuclease I-SceI (S). The TK gene is referred to as “recipient” since it is intended to receive information in the recombinational repair of an I-SceI-induced DSB. Some substrates also contain a TK gene sequence inserted between two HindIII (H) sites to act as a genetic donor in recombinational DSB repair. In all substrates, the orientation of the TK gene coding sequences is from left to right. All constructs also contain the neo gene, transcribed from right to left. (B) Schematics of specific DNA substrates. Only recipient and donor (if any) TK gene sequences are shown for each substrate, but all DNA constructs have the general configuration shown in panel A. The 360-bp donor TK gene sequence on pTKSce-360 has 5′ and 3′ truncations of coding sequences. The 2-kb donor sequence on pTKSce-26 contains a complete TK gene disrupted by an 8-bp XhoI linker insertion (X). Indicated on the two donor sequences is the position of the SstI site (Ss) corresponding to the site into which the I-SceI oligonucleotide was inserted in the recipient TK gene sequence. The 2.5-kb recipient sequence on pTKSce2 is disrupted by a 47-bp oligonucleotide containing two I-SceI sites flanking an XbaI (Xb) site. See Materials and Methods for further details.
FIG. 2
FIG. 2
Intrachromosomal DSB repair products generated in the absence of a homologous donor sequence. Presented at the top of the figure is the parental DNA sequence of the I-SceI recognition site insertion (uppercase letters) within the TK gene (lowercase letters) on pTKSce (Fig. 1B). Sites of staggered cleavage by I-SceI are indicated by arrowheads, and the 4-bp repeats flanking the I-SceI site are underlined. Presented below the parental sequence are nucleotide sequences determined for products of intrachromosomal DSB repair recovered from 51 independent HAT-resistant clones derived from cell line Sce-3 following the introduction of I-SceI into cells. All repair products displayed small deletions ranging from 1 to 22 bp that restored the TK gene reading frame. No., number of HAT-resistant clones.
FIG. 3
FIG. 3
Intrachromosomal NHEJ products generated in the presence of a 360-bp or 2.0-kb homologous donor. Presented at the top of the figure is the parental DNA sequence of the I-SceI recognition site insertion (uppercase letters) within the recipient TK gene (lowercase letters) on pTKSce-360 and pTKSce-26 (Fig. 1B). Sites of staggered cleavage by I-SceI are indicated by arrowheads, as are the 4-bp repeats flanking the I-SceI site (underlining). Presented below the parental sequence are nucleotide sequences determined for the products of intrachromosomal NHEJ products recovered from 6 independent HAT-resistant clones derived from cell lines 360-1 and 360-2 and from 11 independent HAT-resistant clones derived from cell lines 26-1 and 26-2 following the introduction of I-SceI into cells. All NHEJ repair products displayed small deletions ranging from 1 to 16 bp that restored the TK gene reading frame. It should be noted (see Table 2 and text) that some HAT-resistant clones derived from lines 26-1 and 26-2 displayed wild-type TK gene sequences as products of HR but are not shown here. No., number of HAT-resistant clones.
FIG. 4
FIG. 4
Representative Southern blotting analysis of HAT-resistant clones derived from cell line 26-2. (A) DNA samples (8 μg each) isolated from HAT-resistant clones were digested with various combinations of BamHI (B), XhoI (X), and SstI (S), as indicated below the lanes, and were displayed on a Southern blot by using a TK gene-specific probe. Digestions from each individual clone are presented in three adjacent lanes (for example, lanes 1 to 3), with the exception of the clone presented in lanes 13 and 14. Clones displayed in lanes 1 to 9, 13, and 14 were recovered from cell line 26-2 following transfection of pCMV-I-SceI into cells. The HAT-resistant clone displayed in lanes 10 to 12 arose spontaneously (following a mock transfection). In total, 42 DSB-induced and 20 spontaneous HAT-resistant clones from cell lines 26-1 and 26-2 were analyzed by Southern blotting. See text for details. (B) DNA samples (8 μg each) from six clones exhibiting an aberrant restriction pattern similar to that exhibited by the clone in lanes 7 to 9 in panel A were digested simultaneously with BamHI and I-SceI and displayed on a Southern blot by using a TK gene-specific probe.
FIG. 5
FIG. 5
Substrate for monitoring precise religation. Shown at the top of the figure is the 47-bp oligonucleotide inserted into the TK gene on pTKSce2. The oligonucleotide causes a frameshift mutation in the TK gene and contains two I-SceI recognition sites (5′-TAGGGATAACAGGGTAAT-3′) flanking an XbaI site (5′-TCTAGA-3′). The actual sites of cleavage by I-SceI and XbaI on the top strand of the construct are indicated. As illustrated, simultaneous cleavage of the substrate at both I-SceI sites followed by loss of the intervening 23-bp fragment and precise religation of the two outermost I-SceI sticky ends produces a functional, and thus recoverable, TK gene containing a 24-bp insert harboring a single I-SceI site.
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References

    1. Bernstein C, Bernstein H. Aging, sex, and DNA repair. San Diego, Calif: Academic Press, Inc.; 1991.
    1. Bollag R J, Waldman A S, Liskay R M. Homologous recombination in mammalian cells. Annu Rev Genet. 1989;23:199–225. - PubMed
    1. Camerini-Otero R D, Hsieh P. Homologous recombination proteins in prokaryotes and eukaryotes. Annu Rev Genet. 1995;29:509–552. - PubMed
    1. Choulika A, Perrin A, Dujon B, Nicolas J-F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol. 1995;15:1963–1973. - PMC - PubMed
    1. Derbyshire M K, Epstein L H, Young C S H, Munz P L, Fishel R. Nonhomologous recombination in human cells. Mol Cell Biol. 1994;14:156–169. - PMC - PubMed

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