Fig 1.
hetDNA patterns predicted for DSB- versus nick/gap-induced COs.
Solid red and black lines represent the strands of broken and intact chromosomes, respectively. The vertical gray lines indicate the position of the initiating break and arrowheads indicate 3′ ends. Dotted lines correspond to nascent DNA, with the color corresponding to that of the template. hetDNA is boxed in yellow and blue triangles represent HJ cleavage sites. (A) In the classic DSB repair model, 5′ to 3′ resection of each end generates single-strand 3′ tails, one of which invades the donor duplex to create a D-loop and a region of hetDNA. DNA synthesis primed from the invading end enlarges the D-loop until it anneals to the other side of the DSB (2nd-end capture), generating a second hetDNA tract and an HJ. Gaps resulting from the initial 5′-end resection are filled prior to cleavage of the HJs by resolvases. There is a hetDNA tract upstream of the DSB in one CO product and a hetDNA tract downstream of the DSB in the other, which is referred to as two-sided asymmetric hetDNA. (B) In the Meselson-Radding model, DNA extension from the 3′ end of a nick displaces a 5′ flap that invades the donor duplex to form a D-loop and a single region of hetDNA. The D-loop is degraded, leading to formation of an HJ. After HJ cleavage, there is a single, asymmetric hetDNA tract within what was originally donor sequence. (C) A nick can be expanded into a gap by an exonuclease, with subsequent formation of adjacent regions of symmetric and asymmetric hetDNA that are flanked by HJs. This hetDNA pattern persists following HJ resolution.
Fig 2.
Crossover-specific assay for hetDNA mapping.
The recipient lys2Δ5′ allele (white box) on chromosome II contains an in-frame I-SceI cut site (arrowheads indicate corresponding nicks). The lys2Δ3′ allele (black box) on chromosome V is also in frame and contains an I-SceI cut site inactivated by a 6-bp insertion (boxed). The nearest flanking SNPs (red) are 8 bp from each 3′ end of the I-SceI generated DSB. Only a CO event can reconstitute a full-length LYS2 allele and both recombination products must co-segregate for Lys+ colony formation. hetDNA flanking the initiating DSB is indicated as gray boxes, and was monitored in each product by SMRT sequencing. The product-specific primers for PCR amplification are indicated by black horizontal arrows.
Fig 3.
DSB-induced COs with two-sided hetDNA.
(A) The lys2Δ5′ and lys2Δ3′ alleles are shown as white and black boxes, respectively, and the SNPs associated with each are shown. The thunderbolt indicates the position of the DSB in the lys2Δ5′ allele. In an idealized DSB-associated CO, the broken white allele receives information from the black allele, generating a single asymmetric hetDNA tract (gray boxes) in each product on opposing sides of the DSB. There is no alteration of the donor black allele. (B) Each line represents the products of individual CO events, with the lys2Δ5′Δ3′ and LYS2 alleles in the left and right panels, respectively. The vertical red lines indicate the position of the DSB. In all events, hetDNA was upstream of the DSB in the lys2Δ5′Δ3′ allele and downstream of the DSB in the LYS2 allele. The three events marked with an asterisk had terminal symmetric hetDNA consistent with migration of one HJ away from the initiating break (see Fig 4B). “n” is the number of events with the two-sided hetDNA pattern.
Fig 4.
Origins of complexities associated with two-sided hetDNA.
Red and black lines represent broken recipient and intact donor sequences, respectively. Yellow boxes correspond to hetDNA and blue-filled triangles to sites of HJ cleavage. The position of the initiating DSB is indicated as a gray, vertical lines. (A) Gap expansion results from loss of the 3′ end of a DSB and produces a gene conversion tract adjacent to the DSB. (B) Branch migration of one HJ away from the DSB creates a tract of terminal symmetric hetDNA. (C) Spontaneous ejection of an extending end from the black donor can be followed by a temporary template switch to the sister chromatid, followed by a final switch back to the donor. This generates an interstitial tract of symmetric hetDNA, the repair of which can result in an interstitial gene-conversion tract.
Fig 5.
DSB-induced COs with one-sided hetDNA.
Each line represents the products of individual CO events, with the lys2Δ5′Δ3′ allele on the left and the LYS2 allele on the right; red vertical lines correspond to the position of the initiating DSB. Events with hetDNA limited to the (A) left or (B) right side of the DSB are grouped. (C) The transition of asymmetric hetDNA from one product to the other is displaced from the DSB. “n” is the number of events with the relevant hetDNA pattern.
Fig 6.
Mechanisms that produce hetDNA on only one side of an initiating DSB.
Red and black lines represent recipient and donor sequences, respectively. Yellow boxes correspond to hetDNA and blue-filled triangles to sites of nicking by structure-selective endonucleases. The position of the initiating DSB is indicated by vertical gray lines. (A) Migration of an HJ back to the DSB site prior to second-end extension removes the hetDNA created by the initial strand invasion. (B) Migration of one of the HJs past the site of the DSB after second-end extension places both asymmetric hetDNA tracts on the same side of the break. The transition between the hetDNA tracts remains, but is displaced from break site. (C) The D-loop is nicked prior to extension of the invading end to create a single CO product and a broken arm of the donor chromosome (black). As shown, extension of the 3′ (red) end on the other side of the original DSB is templated from the intact sister chromatid. This provides homology to the broken donor and the reciprocal CO product is generated by an annealing reaction.
Fig 7.
Spontaneous COs in which each product contains hetDNA.
White, black, and gray boxes represent lys2Δ5′, lys2Δ3′ and hetDNA, respectively. Potential initiation sites are marked with vertical red lines. (A) COs with a hetDNA pattern consistent with an initiating DSB in the lys2Δ5′ allele. (B) COs with a hetDNA pattern consistent with an initiating DSB in the lys2Δ3′ allele. (C) COs exhibiting a hetDNA pattern consistent with initiation by a single-strand nick. (D) COs containing a hetDNA pattern consistent with initiation by a single-strand gap. “n” is the number of events with the relevant hetDNA pattern.
Fig 8.
hetDNA profiles of spontaneous COs in a tsa1Δ background.
White, black, and gray boxes represent lys2Δ5′, lys2Δ3′ and hetDNA, respectively. Potential initiation sites are marked with vertical red lines. (A) COs with a hetDNA pattern consistent with an initiating DSB in the lys2Δ5′ allele. (B) COs with a hetDNA pattern consistent with an initiating DSB in the lys2Δ3′ allele. (C) COs exhibiting a hetDNA pattern consistent with initiation by a single-strand nick. (D) COs containing a hetDNA pattern consistent with initiation by a single-strand gap. “n” is the number of events with the relevant hetDNA pattern.
Fig 9.
Distribution of hetDNA associated with COs and SDSA-type NCOs.
The distances of SNPs from the DSB site (position 0; negative numbers are for upstream distances) are shown on the x-axis and the percentages of repair events with hetDNA that extends a given distance are on the y-axis. CO-associated hetDNA length distributions on each side of the DSB (blue lines) are from the current data (see S1 Appendix). The NCO-associated hetDNA length distribution (red line) on the downstream side of the DSB was previously reported [36]. Median hetDNA lengths are indicated.