Infrequent co-conversion of markers flanking a meiotic recombination initiation site in Saccharomyces cerevisiae

Abstract

To study the mechanism of meiotic recombination in Saccharomyces cerevisiae, we examined recombination in an interval where the majority of events are initiated at a single hotspot for DNA double-strand breaks (DSBs), with little or no expected contribution by outside initiation events. This interval contained infrequently corrected palindromic markers 300 bp to the left and 600 bp to the right of the DSB hotspot. Conversion of single markers occurred frequently, while conversion of both markers occurred rarely, and many of the tetrads in which both markers converted were the products of multiple events. These data indicate that most meiotic recombination intermediates are asymmetrically positioned around the initiating DSB, with a short (<300 bp) tract of heteroduplex DNA (hDNA) to one side and hDNA on the other side frequently extending 600 bp or more. One consequence of this asymmetry is the preferential concentration of crossovers in the vicinity of the initiating DSB.

Figure 1.—

DSBR model for meiotic recombination (Szostak et al. 1983; Sun et al. 1991). DSBs form and are resected to give 3′ single-strand tails. These tails invade homologous sequences and initiate DNA synthesis (broken lines) to form a dHJ intermediate. Both NCO and CO products are formed by junction cleavage; in every case, the two products contain hDNA on opposite sides of the DSB.

Figure 2.—

The recombination assay systems used in this study. (A) The 3.5-kb URA3-ARG4 insert. Coding sequences are represented by gray and black boxes and noncoding sequences are represented by gray and black lines. Inserted between URA3 and ARG4 is 65 bp of telomeric DNA (TEL, diagonally hatched box) that creates a strong meiotic DSB hotspot (see text) indicated by an arrow. Palindromic markers (lollipops) are inserted at +104 of the URA3 open reading frame and at +9 of the ARG4 open reading frame. These markers are ∼200–340 and 520–660 bp from the center of the DSB hotspot, respectively. (B) Ectopic recombination system. The URA3-ARG4 interval was inserted at HIS4 (box with wavy hatching) on one copy of chromosome III and at LEU2 (checkered box) on the homolog. The two insert sites at HIS4 and LEU2 are 17.9 kb apart in the SK1 strains used here. The marker configuration shown here is present in strain MJL2902, where the insert at HIS4 contains ura3-EcPal+104 and arg4-EcPal+9. In strain MJL2870, the insert at LEU2 contains arg4-EcPal+9 and the insert at HIS4 contains ura3-EcPal+104. (C) Allelic recombination was examined between URA3-ARG4 intervals inserted at HIS4 on both copies of chromosome III. Homologs are distinguished by natMX (horizontally hatched box) or hphMX (vertically hatched box) insertions, ∼4.4 and 8.4 kb from the center of the DSB hotspot, respectively.

Figure 3.—

DSBs in the URA3-ARG4 interval. (A and B) Symbols are as in Figure 2. Southern blot of DNA isolated from a meiotic culture of MJL2961, a rad50S strain homozygous for the insertion at HIS4. (A) DNA from 0 and 7 hr after induction of meiosis was digested with XmnI (X) and probed with ARG4 sequences as described in materials and methods. Size markers (lane not shown) are a BstEII digest of bacteriophage λDNA. (B) For higher-resolution mapping of the DSB hotspot, DNA was digested with PpuMI (P) and BanII (B) and probed as described in materials and methods. Arrows indicate the location of DSBs. Size markers (lane not shown) are MseI (728 bp), MspAI (665 bp), and EarI (278 bp) digests of DNA from a mitotic culture of S1898 (see materials and methods). The 862-bp size marker is a BanII/PpuMI digest of 0 hr DNA of MJL2961. (C) Pulsed-field gels of undigested DNA from meiotic cells, using electrophoresis conditions and a probe (YCL075w) to reveal DSBs on the left arm of chromosome III. (Top lane) Strain MJL2961; (bottom lane) strain MJL3010. Traces are of phosphorimager signals for the relevant portion of each lane (black line, MJL2961; gray line, MJL3010); peak intensities of the full-length chromosome III bands (marked with asterisks) are 2.1 × 10³ psl and 1.9 × 10³ psl for MJL2961 and MJL3010, respectively (psl are units that represent phosphor signal intensity). About 30% of chromosomes III suffer breaks in the left arm in both MJL2961 and MJL3010; in MJL2961, two-thirds of these breaks map to the his4::URA3-ARG4 insert. The trace for MJL3010 is broken to account for the absence of an insert at HIS4. The position of the recombination interval relative to the centromere is indicated on the schematic; size markers (lane not shown) were a HindIII digest of bacteriophage λDNA and λDNA concatamers.

Figure 4.—

Position of the exchange point among CO products. Symbols are as in Figure 2. (A) In the ectopic recombination system, the exchange point can be located in one of three intervals defined by flanking heterology (wavy and checkered boxes) and by the two palindromic markers (lollipops). The size (in kilobases) and frequency of exchanges mapped to the interval (percentage of crossover tetrads) are given below the schematic; crossover intensity (centimorgans per kilobase) in each interval is given in the bar graph above. The average crossover intensity for chromosome III is 0.48 cM/kb (Cherry et al. 2002). Values reflect combined data from MJL2902 and MJL2870. The frequency of exchange (crossover tetrads/total tetrads) in intervals I, II, and III is 3%, 14%, and 8% for MJL2902 and 3%, 17%, and 7% for MJL2870. An additional 3% of combined tetrads had an exchange that could have occurred in either interval I or interval II; an additional 4% of combined tetrads had an exchange that could have occurred in either interval II or interval III. (B) In the allelic recombination system, intervals I and III are subdivided by restriction enzyme site polymorphisms for EcoRI (E) and SphI (S) as described in the text. “Genetic” refers to the frequency of tetrads with exchange that mapped to intervals I, II, and III; values reflect combined data from MJL2936 and MJL2957. An additional 5% of tetrads had an exchange that could have occurred in either interval I or interval II, and an additional 3% of tetrads had an exchange that could have occurred in either interval II or interval III. “Molecular” refers to the frequency of tetrads expected to have an exchange in the subintervals Ia, Ib, IIIa, and IIIb, as calculated from physical analysis of DNA from random spores (see text). The bar graph shows the crossover intensity (centimorgans per kilobase) in each of the five intervals. Values for intervals Ia, Ib, IIIa, and IIIb were calculated from molecular data and, for interval II, from tetrad analysis. (C) Physical analysis of DNA isolated from either nourseothricin-resistant (NOU) or hygromycin-B-resistant (HYG) spore colonies of MJL2976. The schematic represents the marker configuration in this strain: one of the homologs is marked by natMX (horizontally hatched rectangle) and contains arg4-EcPal+9 at HIS4. The other homolog is marked by hphMX (vertically hatched rectangle). Intervals Ib and II cannot be distinguished because ura3-EcPal+104 is absent. DNA was isolated from a pool of at least 200 colonies, digested with EcoRI (E) and SphI (S), and fragments were probed for ARG4 sequences downstream of the palindromic marker. From nourseothricin-resistant spore colonies of MJL2976, the 9.3-kb fragment results from COs with the exchange point in interval Ia; the 6.6-kb fragment is from an exchange in interval Ib, II, or gene conversion of arg4-EcPal+9 associated with an exchange in interval IIIa; the 4.7-kb fragment results from COs with an exchange point in interval IIIa; the 4.4-kb band is the NCO product; and the 2.6-kb band is the parental fragment. From the hygromycin-B-resistant colonies, the 9.3-kb band is the parental fragment; the 6.6-kb band results from exchanges in interval Ib, II, or gene conversion of arg4-EcPal+9 associated with an exchange in interval IIIa; the 4.7-kb band is from COs with exchange in interval IIIa or gene conversion of the SphI site; and the 2.6-kb band is the result of COs with exchange in interval IIIb. The source of the contaminant (?) in the NOU lane is unknown. (D) Physical analysis of DNA isolated from either nourseothricin-resistant or hygromycin-B-resistant spore colonies of MJL2959, analyzed as in C. This strain contains ura3-EcPal+104 at HIS4 on the homolog marked with natMX. Intervals II and IIIa cannot be distinguished because arg4-EcPal+9 is absent. From nourseothricin-resistant spore colonies, the 9.3-kb fragment results from exchanges in interval Ia; the 6.6-kb band is the result of exchanges in interval Ib; the 5.6-kb band results from exchanges in II, IIIa, or gene conversion of the SphI site; the 4.4-kb band is the NCO product; and the 2.6-kb band is the parental fragment. From the hygromycin-B-resistant colonies the 9.3-kb band is the parental fragment; the 7.1-kb fragment results from gene conversion of the SphI site; the 6.6-kb band results from gene conversion of ura3-EcPal+104 associated with exchange in intervals IIIa and II or from a CO in interval Ib; the 5.6-kb band results from gene conversion of ura3-EcPal+104 exchanges in intervals II or IIIa; and the 3.5-kb band results from exchanges in interval IIIb.

Figure 5.—

Resolution of the dHJ intermediate. The intermediate diagrammed here is for the marker configuration in MJL2902, or the allelic cross, and can be resolved in two ways to give rise to CO-associated gene conversion of arg4-EcPal+9. Type 1 resolution (open arrowheads) cuts the HJ on the continuous stands that do not contain newly synthesized DNA and leads to a DSB-distal exchange to the right of arg4-EcPal+9. Type 2 resolution (solid arrowheads) cuts the HJs on the strands containing newly synthesized DNA and their equivalent strands on the other duplex leading to a DSB-proximal exchange to the left of arg4-EcPal+9. “Remote” refers to tetrads where an unconverted palindromic maker separates the crossover from the palindromic marker in hDNA. The table lists the number of tetrads with unidirectional PMS conversion for either ura3-EcPal+104 or arg4-EcPal+9 with type 1, type 2, or remote resolution. The solid and open circles represent the genotype of the tetrad colonies where the parental configuration was ○○○○/••••. Circles that are half-white represent PMS. X marks the exchange point.

Figure 6.—

Models for meiotic recombination based on our findings as well as those of others. (A) Extensive hDNA is formed by extension of the single-strand tail and second end capture. One end of the resected DSB invades homologous sequences forming a short (<300 bp) tract of hDNA and initiates DNA synthesis. NCO products arise through a SDSA pathway where the newly synthesized DNA is displaced and anneals to the other end of the DSB. A final round of DNA synthesis completes the mature NCO product. If the second end of the DSB is captured prior to strand displacement, a dHJ is formed. This intermediate is resolved by the two HJs in opposite directions (open arrowheads) to form CO products; only one of the two resolution modes is shown. A minor pathway for NCO production by unwinding the dHJ intermediate cannot be excluded. (B) Extensive hDNA is formed during initial strand invasion. One of the resected ends invades the homolog to form a tract of hDNA >600 bp. Capture of the second end of the resected DSB leads to formation of the dHJ that is resolved to form CO products as described above. Displacement of the newly synthesized strand in the intermediate shown in step 2 does not form an NCO gene conversion product.