Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions

Abstract

The Rad2/XPG family nuclease, Exo1, functions in a variety of DNA repair pathways. During meiosis, Exo1 promotes crossover recombination and thereby facilitates chromosome segregation at the first division. Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs). Nucleolytic resection of DSBs generates long 3' single-strand tails that undergo strand exchange with a homologous chromosome to form joint molecule (JM) intermediates. We show that meiotic DSB resection is dramatically reduced in exo1Δ mutants and test the idea that Exo1-catalyzed resection promotes crossing over by facilitating formation of crossover-specific JMs called double Holliday junctions (dHJs). Contrary to this idea, dHJs form at wild-type levels in exo1Δ mutants, implying that Exo1 has a second function that promotes resolution of dHJs into crossovers. Surprisingly, the dHJ resolution function of Exo1 is independent of its nuclease activities but requires interaction with the putative endonuclease complex, Mlh1-Mlh3. Thus, the DSB resection and procrossover functions of Exo1 during meiosis involve temporally and biochemically distinct activities.

Figure 1. Analysis of Meiotic DSB Resection

(A) Structure of the HIS4LEU2 locus showing open-reading frames and diagnostic XhoI restriction sites (circled X’s). The DSB fragments released by XhoI digestion and locations of probes for Southern analysis are shown below. (B) Diagram of DSB component-strands analysis showing the signals detected by Southern analysis of a native/denaturing 2D gel hybridized with probes to both strands. The right-hand panels show Southern analysis of DSBs from wild-type cells, successively hybridized with probes to both strands and to individual strands. (C-D) Southern analysis of DSB resection over time in wild-type and exo1Δ meiotic time-courses. (D) Quantification of the resection profiles of DSBs in wild-type and exo1Δ meiotic cultures sampled at 4 hrs. (E) Quantification of the resection profiles of DSBs in wild-type and exo1Δ meiotic cultures sampled at 4 hrs. (F) Quantification of the resection profiles of DSBs in pCLB2-SGS1 and pCLB2-SGS1 exo1Δ meiotic cultures sampled at 5 hrs. (G) Southern images of DSB resection in pCLB2-SGS1 and pCLB2-SGS1 exo1Δ mutants.

Figure 2. Physical Analysis of Meiotic Recombination in Wild-Type and exo1Δ Cells

(A) Map of the HIS4LEU2 locus showing diagnostic XhoI restriction sites (circled X’s) and the position of the probe. DNA species detected with Probe 4 are shown below. (B) Southern images of 1D gels hybridized with Probe 4 showing DNA species detailed in (A). (C) Quantitative analysis of DSBs, crossovers and meiotic divisions (MI ± MII). % DNA is percent of total hybridizing DNA. MI ± MII is cells that have completed either the first or second meiotic divisions as detected by the number of DAPI-staining bodies. (D) Graphs representing DSBs and crossovers normalized to the internal maximum.

Figure 3. Analysis of Joint Molecule Formation in Wild-Type and exo1Δ Cells

(A) Map of HIS4LEU2 locus showing diagnostic restriction sites and the position of the probe. JM species detected with Probe 4 are shown below. Presumed structures of SEIs and dHJs are shown in the left-hand panel. SEI, single-end invasion; IH-dHJ, interhomolog double Holliday Junction; IS-JM, intersister-joint molecule; mcJM, multi-chromatid joint molecule. mcJMs comprise either three or four interconnected chromatids; chromatid composition of mcJMs are shown in parentheses, e.g. MDD = 1 Mom + 2 Dad chromatids. (B) Southern blot of native/native 2D gel showing JMs detailed in (A). (C) 2D analysis of JMs in wild-type and exo1Δ cells. JM regions are magnified in the right-hand panels. (D) 2D analysis of JMs in ndt80Δ and ndt80 exo1Δ cells. (E) Quantification of JMs in wild-type and exo1Δ cells. % DNA is percent of total hybridization signal. (F) Quantification of JMs in ndt80Δ and ndt80Δ exo1Δ cells.

Figure 4. Analysis of DSB Resection and Meiotic Recombination in exo1-D173A Cells

(A) Domains of the Exo1 polypeptide showing conserved N (N-terminal) and I (internal) nuclease domains, and position of the D173A allele (asterisk). (B) Comparison of DSB resection profiles in exo1Δ and exo1-D173A cells. The right-hand panels show representative images of 2D native/denaturing gels hybridized with the 5′ bottom probe. (C) Genetic analysis of crossing over in wild-type, exo1D, and exo1-D173A cells. Intervals analyzed by tetrad analysis, and graphs of map distances (±SE) and spore viability, are shown. Asterisks indicate significant differences between map distances p < 0.005 (see Table S1). cM, centiMorgans. (D) Physical analysis of recombination in wild-type, exo1Δ and exo1-D173A cells. Images of 1D Southern analysis, and quantitative analysis of DSBs, crossovers and meiotic divisions (MI ± MII). (E) Final crossover levels in wild-type, exo1Δ and exo1-D173A cells. Graphs show the averages of three independent time courses (means ± S.E.).

Figure 5. Crossing-Over Along Chromosome III in Wild-type and exo1-D173A Cells

(A) Genetic intervals on chromosome III analyzed by tetrad analysis. (B and C) Map distances (±SE) in wild-type and exo1-D173A cells. Asterisk indicates significantly different by G-test, p = 8 × 10-5 (see Table S2).

Figure 6. The Crossover Function of Exo1 Involves Interaction With Mlh1

(A) Domain structure of EXO1 and the four alleles analyzed. (B) Physical analysis of crossing-over in exo1 mutant strains. (C) Quantitative analysis of crossing-over in exo1 mutant strains. For each strain, at least three independent time-course experiments were analyzed (bars show the mean value ± S.E. for the 13 hr time-points, when crossover levels plateau). (D) Analysis of DSB resection in wild-type and exo1-FF477AA cells. (E) Space filling model of Mlh1 showing the position of the S2 interaction site and E682 residue. The S1 site mediates heterodimerization with the other MutL homologs, Pms1, Mlh2 and Mlh3. (F) Physical analysis of crossing-over in wild-type, mlh1-E682A and mlh1Δ cells. (G) Quantitation of crossover levels in wild-type, mlh1-E682A and mlh1Δ cells. Graphs show the averages of at least three independent time courses (means ± S.E. for the 13 hr time-points). (H) Physical assay to detect unrepaired heteroduplex DNA. (I) Analysis of heteroduplex repair in wild-type, mlh1-E682A and mlh1Δ cells. (J) Quantitation of heteroduplex levels in wild-type, mlh1-E682A and mlh1Δ cells. Graphs show the averages of at least three independent time courses (means ± S.E. for the 13 hr time-points).

Figure 7. Models of Exo1 Function During Meiotic Recombination

(A) Three models for meiotic DSB-resection (see text for details). (B) Nuclease-independent function of Exo1 in resolving dHJs into crossovers (see text for details).