Expansions and contractions in a tandem repeat induced by double-strand break repair - PubMed (original) (raw)

Expansions and contractions in a tandem repeat induced by double-strand break repair

F Pâques et al. Mol Cell Biol. 1998 Apr.

Abstract

Repair of a double-strand break (DSB) in yeast can induce very frequent expansions and contractions in a tandem array of 375-bp repeats. These results strongly suggest that DSB repair can be a major source of amplification of tandemly repeated sequences. Most of the DSB repair events are not associated with crossover. Rearrangements appear in 50% of these repaired recipient molecules. In contrast, the donor template nearly always remains unchanged. Among the rare crossover events, similar rearrangements are found. These results cannot readily be explained by the gap repair model of Szostak et al. (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) but can be explained by synthesis-dependent strand annealing (SDSA) models that allow for crossover. Support for SDSA models is provided by a demonstration that a single DSB repair event can use two donor templates located on two different chromosomes.

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Figures

FIG. 1

The SDSA model, and how it could explain rearrangements in tandem arrays. (A) Two alternative DSB repair models. The ends of the break are first resected (step 1), and the resulting 3′ ends can invade a homologous template and prime DNA synthesis (steps 2 and 3). Szostak et al. (68) proposed that resolution would require cutting of two Holliday junctions, resulting in crossover or noncrossover products (step 4), but to explain the lack or low frequency of crossover events accompanying mitotic gene conversion, other authors (10, 22, 42) proposed that the newly synthesized strands could be unwound from the template and anneal together (step 5). (B) A model of tandem repeat rearrangements derived from the DSB repair model of Szostak et al. (68). Expansions and contractions could be due to slippage events during semiconservative DNA synthesis (step 1). The resulting loop could be corrected in either direction, or not be corrected, but anyway, after resolution of the Holliday junctions (step 2), the rearrangements should be found on both donor and recipient molecules. (C) Origin of tandem repeat rearrangements according to a simple SDSA model. Both 3′ ends of the broken molecule invade the homologous template and prime DNA synthesis (step 1). Then, the newly synthesized strands are unwound from the template and anneal together. Two of the many possibilities of annealing are represented (step 2).

FIG. 2

Experimental system to study DSB repair involving tandem arrays. An HO cut site is introduced into a _Kpn_I site of the chromosomal endogenous LEU2 gene. The HO gene can be expressed from an inducible promoter and will cut the LEU2 gene (A). The structures of the templates are shown as follows: no homologous template (B); plasmid LEU2 template (C); plasmid LEU2 templates containing eight D. melanogaster 5S genes in a tandem array (D), two 5S arrays surrounding the D. melanogaster white gene (E), the 5′ part of the white gene (F), or the entire white gene (G); chromosomal LEU2 template (H); and chromosomal leu2 template with a 9.1-kb insert corresponding to two 5S arrays surrounding the white gene (I). Shaded box, LEU2 gene; open box, one D. melanogaster 5S gene (375 bp); solid box, D. melanogaster white gene; open circle, centromere. The number of repeats in each 5S array is given above the array.

FIG. 3

Recombinants obtained by DSB repair involving tandem repeats. For each panel, the structure of the template is diagrammed in the upper box, and the numbers of recipient (R) and donor (D) molecules with each structure are given on the right. (A) Forty-four recombinants obtained after DSB repair using the plasmid template diagrammed in Fig. 2D. (B) Forty recombinants obtained after DSB repair using the plasmid template diagrammed in Fig. 2E. (C) Thirty-four noncrossover recombinants obtained after repair using the template diagrammed in Fig. 2I. (D) Forty-one noncrossover recombinant lines obtained after repair in G1 of the template diagrammed in Fig. 2I. In seven cases (bottom), two populations of cells differing in the structure of the repaired molecule, were obtained. In panels B and D, one recombinant displays tandem repeat rearrangement in both donor and recipient. In these cases, the corresponding donor and recipient molecules are labeled with stars.

FIG. 3

FIG. 4

Crossover events. The diagram at the top represents the donor and the recipient. The cutting of the donor by HO endonuclease, and genetic and molecular identification of crossover events (with _Sph_I and _Eag_I diagnostic restriction enzymes) allowed us to characterize the 29 events whose structures are shown here.

FIG. 5

A model of SDSA compatible with crossovers. (A) A single 3′ end invades the template and primes synthesis (step 1) and is extended by bubble migration (step 2). Most of the time, resolution can occur by annealing (step 3). However, invasion of the template by the second 3′ end may sometimes stabilize the strand displaced by the first 3′ end (step 4). DNA synthesis would become semiconservative (step 5), and Holliday junctions could be formed (step 6) and subsequently cut. (B) Creation of tandem repeat rearrangements by reinvasion. A single 3′ end would invade the donor template and initiate DNA synthesis (step 1). It would then be unwound from the template (step 2) and invade this template a second time. Because repeated sequences have been added to the 3′ end, this second invasion can be initiated on any of the tandem repeats on the template. Two different possibilities are shown here (step 3). The conversion could then be accomplished by annealing or by formation and cutting of Holliday junctions, as shown in panel A.

FIG. 6

Direct evidence for the SDSA model. (A) A plasmid with a gapped leu2 gene can be repaired on an uninterrupted LEU2 template. This event minimally requires one strand invasion. The newly synthesized strand can then anneal to the other 3′ end of the DSB. (B) A truncated template with no overlap with one side of the gap does not allow for efficient repair. (C and D) Two truncated templates also allow for gap repair. Each template is lacking an overlap on one side of the gap but is overlapping with the other template. On each panel, an arrow(s) represents the invading 3′ end(s) after it has been extended by DNA synthesis. Panels C and D represent two different mechanisms to account for the same events: both 3′ ends invade and prime DNA synthesis before the newly synthesized DNA strands are unwound from the templates and anneal together (C), or DNA synthesis from a single 3′ end can switch from one template to the other (D). In both cases, two strand invasion steps and one annealing step are required.

References

1. Alani E, Cao L, Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987;116:541–545. - PMC - PubMed
1. Armour J A, Jeffreys A J. Biology and applications of human minisatellite loci. Curr Opin Genet Dev. 1992;2:850–856. - PubMed
1. Bremer M C, Gimble F S, Thorner J, Smith C L. VDE endonuclease cleaves Saccharomyces cerevisiae genomic DNA at a single site: physical mapping of the VMA1 gene. Nucleic Acids Res. 1992;20:5484–5490. - PMC - PubMed
1. Buard J, Jeffreys A J. Big, bad minisatellites. Nat Genet. 1997;15:327–328. - PubMed
1. Canceill D, Ehrlich S D. Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli. Proc Natl Acad Sci USA. 1996;93:6647–6652. - PMC - PubMed

Expansions and contractions in a tandem repeat induced by double-strand break repair - PubMed (original) (raw)