Analysis of repair mechanism choice during homologous recombination

Abstract

Double-strand breaks (DSBs) occur frequently during cell growth. Due to the presence of repeated sequences in the genome, repair of a single DSB can result in gene conversion, translocation, deletion or tandem duplication depending on the mechanism and the sequence chosen as partner for the recombinational repair. Here, we study how yeast cells repair a single, inducible DSB when there are several potential donors to choose from, in the same chromosome and elsewhere in the genome. We systematically investigate the parameters that affect the choice of mechanism, as well as its genetic regulation. Our results indicate that intrachromosomal homologous sequences are always preferred as donors for repair. We demonstrate the occurrence of a novel tri-partite repair product that combines ectopic gene conversion and deletion. In addition, we show that increasing the distance between two repeated sequences enhances the dependence on Rad51 for colony formation after DSB repair. This is due to a role of Rad51 in the recovery from the checkpoint signal induced by the DSB. We suggest a model for the competition between the different homologous recombination pathways. Our model explains how different repair mechanisms are able to compensate for each other during DSB repair.

Figure 1.

Schematic representation of the different DSB repair models. In the DSBR model (2), a double Holliday junction (HJ) can be resolved by endonucleolytic cleavage (indicated by triangles) to generate crossover or gene conversion (non-crossover) products. In the SDSA model (3), D-loop extention and invading strand displacement produce a GC product. In the BIR model (5,6), the invading strand continues DNA synthesis to the end of the DNA molecule, producing a duplication of the chromosome arm. In the SSA model (4), a DSB made between direct repeats results in deletion of one of the repeats and the intervening DNA.

Figure 2.

Repair choice in a strain which undergoes a DSB. (A) Schematic representation of the yeast strain NA14 (DSB within one of intrachromosomal repeats) and its repair products following induction in YEP–Gal medium. (B) Schematic representation of strain NA15 (DSB between intrachromosomal repeats) and its repair products. (C) Graphic representation of the distribution of repair products in NA14 wt, Δrad1, Δrad51 and Δrad1Δrad51 strains. (D) Graphic representation of the distribution of repair products in NA15 wt, Δrad1, Δrad51 and Δrad1Δrad51 strains. Column height represents the viability on YEP–Gal medium compared to YEPD medium. Each column shows the percentage of the deletion events (gray) and GC events (black).

Figure 3.

Graphic representation of the distribution of repair products in different strains. (A) Distribution of intra- (white) and inter-chromosomal events (black) in strains harboring an ectopic/allelic donor of increasing length in wt background. (B) Distribution of intra- (white) and inter-chromosomal events (black) in strains harboring an increasing distance between the intrachromosomal repeats in wt background. (C) Distribution of GC events (black) and deletion events (gray) in strains harboring an increasing distance between the intrachromosomal repeats in wt background. (D) Graphic representation of the repair efficiency of wt and Δrad51 strains with an increasing distance between intrachromosomal repeats.

Figure 4.

Analysis of repair kinetics in Δrad51 strains. Repair kinetics was analyzed in yeast strains carrying intrachromosomal repeats 3 kb apart (NA14Δrad51) or 6.5 kb apart (NA3Δrad51). (A) Quantative PCR measuring the relative amount of deletion product at timely intervals following induction of DSB. The picture on the left depicts the assay conditions. The graph below shows the quantification of the deletion product normalized by the control (STE4) band. (B) Graphic representation of the fraction of large budded cells (up) and FACS analysis (bottom) of cells following induction of DSB. (C) Upper panels show a western blot analysis of the activation kinetics of the Rad53 kinase in NA14Δrad51 and NA3Δrad51. The lower panel shows controls: No protein in a Δrad53sml1-1 strain, MMS- treated/untreated wild-type and Δmec1Δsml1 strains. The phosphorylated form of Rad53 (Rad53-P) migrates more slowly than the un-phosphorylated form (Rad53). Asterisk marks the location of a non-specific band.

Figure 5.

A model for repair pathway choice. (A) Repair choice in a strain where the break is located inside one of the intrachromsomal repeats (NA14). Following DSB formation resection is carried out at both sides of the break by a complex recruited to the break (depicted as a pink circle around the lesion). Among these proteins there is a helicase/nuclease that processes the ends and extrudes ssDNA out to allow homology search. The ssDNA exposed following resection is covered by several proteins such as RPA, Rad51 and checkpoint components (yellow circles). (B) The zoom-in image shows that while resection is carried out base pairing is examined in the repair complex to find homology between the resected strands (homology in cis), while inter- and intra-chromosomal homology is searched by the ends (homology in trans). (C) Repair choice in a strain where the break is located between the intrachromsomal repeats (NA15). Resection and homology search take place as in (A). The end products of the repair is determined by resection reaching intachromosomal homology first.