Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination

Abstract

The molecular structures of crossover (CO) and noncrossover (NCO) intermediates were determined by sequencing the products formed when a gapped plasmid was repaired using a diverged chromosomal template. Analyses were done in the absence of mismatch repair (MMR) to allow efficient detection of strand-transfer intermediates, and the results reveal striking differences in the extents and locations of heteroduplex DNA (hDNA) in NCO versus CO products. These data indicate that most NCOs are produced by synthesis-dependent strand annealing rather than by a canonical double-strand break repair pathway and that resolution of Holliday junctions formed as part of the latter pathway is highly constrained to generate CO products. We suggest a model in which the length of hDNA formed by the initiating strand invasion event determines susceptibility of the resulting intermediate to antirecombination and ultimately whether a CO- or a NCO-producing pathway is followed.

Figure 1

SDSA and DSBR models of gap repair. Solid blue and orange lines correspond to single strands of the gapped and intact alleles, respectively; arrowheads represent the 3′ ends of DNA strands. Dotted lines correspond to new DNA, which is colored according to the template directing its synthesis. The solid triangle at the distal end of the D-loop formed in step A represents the position of Rad1-Rad10 cleavage to facilitate second-end capture and generate an intermediate with a single HJ. With the double HJ, a CO results when the exchanged strands are nicked at one junction and the unexchanged strands are nicked at the other junction; the CO position occurs at the junction where the unexchanged strands are nicked. Gray boxes indicate the positions of hDNA in intermediates and products.

Figure 2

Gap-repair system. The plasmid contains an ARS, the URA3 gene (gray) and a gapped his3 allele (orange). The his3Δ3′ allele is (blue) located at the CAN1 locus on chromosome V. NCO and CO events generate His⁺ transformants with an unstable and stable Ura⁺ phenotype, respectively. The positions of primers used to selectively amplify recombination products are indicated.

Figure 3

NCO products generated in the absence of MMR. The chromosomal his3Δ3′ and gapped alleles are indicated at the top of each panel in blue and yellow, respectively. Gap positions are indicated by the long, vertical blue lines; the positions of silent sequence polymorphisms are indicated by the short, vertical black lines in the gapped alleles (see Figure S1 for sequence changes). Each horizontal line corresponds to an independent gap-repair event, and the extent of DNA transferred from the chromosome is indicated. Green lines correspond to hDNA and blue lines to gene conversion events. Tract endpoints were placed between the most gap-distal polymorphism transferred and the next, unchanged polymorphism.

Figure 4

CO products generated in the absence of MMR. The cartoon above the sequenced events illustrates the products expected if the gap is repaired but no additional flanking sequence is transferred. Plasmid sequence is in yellow, chromosomal sequence in blue and hDNA in green. Classes 1–4 are described in the text.

Figure 5

Relationship between the end that invades the donor, the CO position relative to the gap, and the location of hDNA in CO products. Dotted lines represent DNA synthesized during gap repair and are colored according to the template. Arrowheads represent the 3′ ends of DNA strands and gray boxes highlight the positions of hDNA. In Panel A, the donor allele is invaded by the 3′ end upstream of the gap, and intermediates with one or two HJs are shown. The single HJ postulated to result from D-loop nicking should always be upstream of the gap, as shown. Panel B illustrates nick-directed cleavage of single or double HJs. Filled circles correspond to 5′ ends at nicked HJs and the dashed black arrows indicate the positions where a nicked HJ is predicted to be cleaved by Mus81-Mms4. Such cleavage generates exclusively CO products. See Figure S2 for gene conversion tracts predicted if segregated hDNA fails to be detected.

Figure 6

NCO and CO products of the BglII system in the presence of MMR. See legends to Figures 3 and 5 for NCO and CO details, respectively.

Figure 7

Model relating end resection to SDSA-DSBR pathway choice and MMR-directed antirecombination. Dotted lines represent new DNA, which is color-coded to reflect the template; gray donuts near the extended 3′ ends represent PCNA molecules. Opposed orange and blue triangles represent mismatches in hDNA; gray octagons correspond to bound MMR proteins; and repaired hDNAs (gene conversion tracts) are highlighted by gray boxes. See text for details.