Synthesis-dependent strand annealing in meiosis

Abstract

Recent studies led to the proposal that meiotic gene conversion can result after transient engagement of the donor chromatid and subsequent DNA synthesis-dependent strand annealing (SDSA). Double Holliday junction (dHJ) intermediates were previously proposed to form both reciprocal crossover recombinants (COs) and noncrossover recombinants (NCOs); however, dHJs are now thought to give rise mainly to COs, with SDSA forming most or all NCOs. To test this model in Saccharomyces cerevisiae, we constructed a random spore system in which it is possible to identify a subset of NCO recombinants that can readily be accounted for by SDSA, but not by dHJ-mediated recombination. The diagnostic class of recombinants is one in which two markers on opposite sides of a double-strand break site are converted, without conversion of an intervening heterologous insertion located on the donor chromatid. This diagnostic class represents 26% of selected NCO recombinants. Tetrad analysis using the same markers provided additional evidence that SDSA is a major pathway for NCO gene conversion in meiosis.

Figure 1. Models for Meiotic Recombination

(A) The dHJ model [6]. Recombination is initiated by a DSB. Ends are resected to form 3′ single-stranded tails. One end invades the intact homolog to form a D-loop that is enlarged by extension of the invading end by DNA synthesis using the intact strand as template. End extension enlarges the D-loop, making it possible for the second end to anneal to the D-loop. After the second end anneals, repair synthesis and ligation forms a dHJ. CO and NCO products arise from the relative orientation of two HJ resolution events. (B) The early crossover decision model [25,26]. Ends are resected, and one DSB end forms a D-loop with its homolog and is then extended by DNA synthesis, as in the dHJ model. If a CO is to be formed, events follow those as in (A). If an NCO is to be formed, after the end is extended, the D-loop is disrupted by displacement of the extended end. The displaced end then undergoes synthesis-dependent strand annealing (SDSA; see text): Repair synthesis and ligation forms an NCO recombinant. As drawn here, SDSA forms only NCOs. A derivative of this model showing how a CO can be associated with SDSA is provided in Figure S3.

Figure 2. The “Ends-Apart” Recombination System

(A) D (“Donor”) and R (“Recipient”) contain heteroalleles of his4 and leu2 genes; vertical lines through the heteroalleles indicate the positions of the restriction site fill-in mutations. Breaksites 1, 2, and K (the breaksite in the KanMX cassette) are indicated. The intensities of these breaksites are: site 1 = 12%, site 2 = 3%, and site K = 1.5%. Distances from site 2 (the breaksite of interest to this study) to each of the heteroallelic mutations are shown. D::KanMX-dup contains a heterologous KanMX cassette inserted between the two heteroalleles, and also contains a duplicated segment of the 5′ end of leu2; the duplicated segment is shown as a red box over an arrowhead. D::KanMX is identical to D::KanMX-dup but does not contain the duplication. Site 2 is located ≤100 bp away from the KanMX insertion point. In R/D diploids, two heterozygous markers flank HIS4::LEU2; an ARG4 cassette (peach/orange) inserted at the LEU2 locus, replacing the LEU2 coding sequence and a URA3 cassette (lavender/purple) inserted at the CHA1 locus. (B) During meiosis, after a break at site 2 on the R chromosome, wild-type sequences from the D chromosome must be used to repair the resected regions of R that had carried the his4B and leu2K mutations. Mutations are shown by yellow x's. (1) The R chromatid is cleaved at site 2, and 5′ ends are resected, leaving 3′ overhanging tails. (2) 3′ tails invade the homolog forming a D-loop, and the mismatch repair (MMR) machinery recognizes mismatches (circled). (3) Mismatches are removed by the MMR machinery. (4) Repair synthesis uses D chromatid information to repair the R molecule to His⁺Leu⁺ prototrophy. (5) Invading ends are displaced and anneal. (6) Repair synthesis and ligation for a HIS4⁺LEU2⁺ chromatid.

Figure 3. Models for Generation of HIS4⁺ LEU2⁺ Recombinants via a Single DSB in the Ends-Apart System

Colors represent the same markers as described in Figure 2. (A) Both ends formed by a DSB on R invade R/D::KanMX-dup. (B) Only one of the two ends invades R/D::KanMX-dup. In (A) and (B), the duplicated segment allows for annealing of two ends, each acquiring sequences from different copies of the duplication, thus making it possible to generate HIS4⁺LEU2⁺ recombinants that lack KanMX. (C) Creation of HIS4⁺LEU2⁺ recombinants in R/D::KanMX. Resolution of NCOs by the SDSA mechanism is only expected to give recombinants that contain KanMX. The basic events common to all three mechanisms (A–C) are: (1) The DSB site between his4 and leu2 on R is cleaved. (2) End invasion. (3) Mismatch repair at the site of his4B and leu2K and repair synthesis to extend ends into or through the duplicated region (in R/D::KanMX-dup), but not all the way across the KanMX gene. (4) D-loop disruption. (5) Opportunity for annealing. Ends that contain complementary sequences can anneal. Ends than cannot anneal may reinvade and undergo further extension until sequences complementary to the partner end are added. (6) Strand annealing of disrupted ends. dHJ formation for ends that remain in D-loops. (7) Repair synthesis and ligation, or HJ resolution.

Figure 4. Genotypes of HIS4⁺LEU2⁺ Recombinants

The numbers given are percentages of double prototrophs with the configuration of markers indicated in the diagram on the left. The R/D::Kan-MX-dup strain was from DKB2558 × DKB2050; R/D::Kan-MX from DKB2564 × DKB2050; and zip1/zip1 R/D::Kan-MX-dup strain from DKB2379 × DKB2983. Experiments were performed on at least three separate cultures.