Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways

Abstract

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

Figure 1. Strand transfers during canonical meiotic DSB repair pathways.

For simplicity, only two homologous DNA molecules are represented, one red and one blue. The 3′ DNA end is identified by an arrow when appropriate. Newly synthesized DNA is represented as a dotted line. After a DSB is made, 5′ to 3′ end resection followed by 3′ end invasion of an intact homoduplex DNA molecule are common steps to all recombination pathways. Three major recombination pathways are distinguished according to the intermediates formed. On the left, after 3′ end invasion, DNA synthesis extends the invading end prior to the dismantling of this evanescent intermediate. The DSB is eventually repaired after annealing of the two ends, and gap fill in (a) (SDSA model, [24]). This pathway generates exclusively NCOs with only one hDNA tract. Both ends of a DSB can formally engage in two independent SDSA reactions, generating also a NCO but with two hDNA tracts distributed on the same chromatid in a trans configuration (b). In the middle, after 3′ end invasion, a stable SEI intermediate is formed and is processed into a double Holliday junction-containing intermediate. dHJ resolution can formally lead to CO if the four nicks (arrowheads) cleave four different DNA strands (e), or NCO if the four nicks affect only two DNA strands (d) (these two pathways illustrate the canonical DSBR model [21]). Note that filled and unfilled arrowheads illustrate the two possible resolutions of the same dHJ. The crossover point is defined by the two vertical arrowheads. The top CO pattern therefore corresponds to resolution by the filled arrowheads, and the bottom CO pattern corresponds to resolution by the unfilled arrowheads. Both COs and NCOs resulting from dHJ resolution present two hDNA tracts distributed on the two non-sister chromatids involved in the repair reaction. Alternatively, a dHJ can be dissolved by the combined action of a helicase and a type I topoisomerase and give rise to a NCO with two hDNA tracts on the same chromatid in a trans configuration (c). On the right, after 3′ end invasion, a less stable intermediate than the aforementioned SEI is formed and contains two nicked HJs (f). Structure-specific endonucleases like Mus81 can process such an intermediate into a CO with two hDNA tracts distributed on the two non-sister chromatids involved in the repair reaction, as for the canonical DSBR model (f). The notation 5:3_5:3* stands for two consecutive half conversions or hDNA tracts with the same global strand asymmetry but with different strand distributions. Trans hDNAs are defined by two such hDNA tracts on the same chromatid, while dHJ resolution leads to two such hDNA tracts on two non-sister chromatids.

Figure 2. Rationale for the identification of the global landscape of COs, NCOs, and hDNAs.

Only one DNA molecule of the S288C (red) and SK1 (blue) strains are represented for simplicity. After induction of meiosis, Spo11 generates a DSB on the SK1 chromosome that is repaired by copying genetic information from the S288C chromosome. This homologous recombination process can give rise to either a NCO or a CO as represented here (black cross) and generates hDNA tracts (DNA segments containing two strands of different parental origins i.e. one SK1 (blue) and one S288C (red) strand here). In the presence of MMR (left part), mismatches present in hDNAs are repaired toward either restorations or full conversions leading to 4∶4 and 6∶2 segregation patterns respectively of the corresponding markers in the hybrid progeny. A full conversion is represented here. In the absence of MMR (right part), mismatches from hDNAs are left unrepaired and the corresponding markers present a 5∶3 segregation pattern as shown here. The asterisk indicates that distinct hDNA tracts generate the same global 5∶3 segregation of nearby markers. hDNAs are homogenized into homoduplex DNAs at the first mitotic division of the spores. Genotyping by DNA hybridization onto Affymetrix tiling arrays of the four (presence of MMR) or eight (absence of MMR) clonal cell populations allows CO and NCO identification from recombination products. hDNA identification is possible only in the absence of MMR by genotyping of the eight populations.

Figure 3. COs and NCOs in a SK1 × S288C hybrid in the presence and absence of MMR.

(A) Mean values of COs (blue dots) and NCOs (red dots) per chromosome out of 7 wild type meioses are represented as a function of chromosome size. Chromosome numbers are indicated under the x axis. Vertical dotted lines represent standard deviations. Linear regression curves are represented, with R² corresponding to the square of Pearson's product-moment correlation coefficient, and p being the probability that R² is equal to zero. (B) Same as in A but for a msh2Δ hybrid. Note that COs data come from three meioses and NCOs data come from two meioses. (C) Mean values of COs (blue bars) and NCOs (red bars) for wild type and msh2Δ hybrids with standard deviations indicated except for msh2Δ NCOs where the deviation to the mean has been indicated. Both CO and NCO numbers are significantly higher in msh2Δ compared to wild type (p<0.05, Wilcoxon test).

Figure 4. Representative classes of NCO- and CO-associated strand transfers and model for meiotic crossover control points.

(A) Representative classes of NCO-associated strand transfers are expressed as percentages of total NCOs. The “SDSA-like” and the “trans” classes are divided in two sub-classes: the canonical sub-classes with the expected patterns from the canonical models (Figure 1a, b and c respectively) and the complex sub-classes with patterns presenting the expected profiles plus additional unpredicted 6∶2 or 4∶4 tracts. The “other” class contains NCOs with strand transfer patterns that cannot be attributed unambiguously to a specific origin. (B) Models for the formation of 4∶4 tracts within trans hDNAs associated to NCOs. During double SDSA, one end invades a non-sister chromatid as expected while the second end first invades the sister chromatid then a non-sister chromatid. Annealing of the two ends leads to the formation of a trans hDNA pattern with a 4∶4 tract in the middle. During dHJ dissolution, an unrepaired nick formed before or during the topological processing of the junction can induce nick translation, which generates a 4∶4 tract. (C) Representative classes of CO-associated strand transfers are presented as percentages of total COs with detectable strand transfer. For the “canonical DSBR” class, the two expected patterns are represented (see Figure 1e). The “hDNA on one chromatid” class and the three sub-classes of “hDNA on two non-sister chromatids” are illustrated by examples of observed patterns. The two “DSBR compatible” sub-classes of hDNA on two non-sister chromatids correspond to situations where the strand transfers from the two non-sister chromatids do not overlap. (D) In addition to the first CO control point before or during the transition between DSB and invasion of the homolog by one end of the DSB, our observations support a model where dHJs can be dissolved into NCOs and therefore constitute a second CO control point. The question marks indicate that except SEIs, no recombination intermediates have been isolated for the other pathways.

Figure 5. Models for meiotic CO formation.

CO formation (I) and CO formation (II) are variations of the canonical models that include ligated and non ligated dHJs respectively. (A) Model for strand transfers on only one chromatid after D-loop and dHJ migration. DNA synthesis primed at the first invading end is coupled with migration of the D-loop in the same way (horizontal green arrows). Migration of the D-loop can partially (not shown) or completely erase the first hDNA formed after strand invasion as shown here. The ligated dHJ can be resolved (a) or can keep migrating away from the invasion point (b and c). dHJ migration within a homoduplex DNA generates an aberrant 4∶4 tract or symmetric hDNA tract (indicated by a star), that corresponds to the region encompassed by the two HJs. If dHJ migration is long enough, it can also create a 4∶4 tract located in between the hDNA formed at the initial Spo11-DSB and the closest HJ. Remarkably, aberrant 4∶4 tracts are almost never detected, suggesting they are very small. This implies that the two HJs are cleaved when they are close to one another, in a one- or two-step process as illustrated here. The grey arrows indicate that the two-step resolution process is speculative and as equally probable as a one-step resolution process of two close HJs. (c) illustrates template switching between non-sister and sister chromatids that generates an alternation in strand transfer on a single chromatid. Importantly, migration of the D-loop and dHJ could also occur in the opposite direction compared to the one presented here, but it would lead to the same outcome and has not been represented for simplicity. (B) Models for asymmetric and complex strand transfers on two non-sister chromatids. Asymmetric positioning of recombination intermediates around the initial Spo11-DSB can generate one long and one short hDNA tracts. The asymmetry can come from either a long DNA end invasion and a short DNA synthesis (d) or a short DNA end invasion and a long DNA synthesis (not shown) . Note that such asymmetry can also affect CO formation as proposed in panel A. After first end invasion and second end capture, panel (d) shows the cleavage of the recombination intermediate by a nuclease such as the structure-specific endonuclease Mus81 to generate a CO. Panel (e), depicts how DNA synthesis initiated at transient nicks present in recombination intermediates combined with HJ migration can lead to the inversion of a 5∶3 hDNA tract into a 3∶5 hDNA causing strand transfers associated with COs to be more complex. DNA is cut close to the HJ resulting from the first end invasion by a nuclease such as the structure-specific endonuclease Mus81. DNA synthesis is subsequently initiated at that nick. The invading strand that has already been used as a template for elongation of the second DSB end is also used as a template from that nick thanks to HJ branch migration. Under this scenario, a 5∶3 segregation tract can be converted into an opposite 3∶5 tract. Note that template switching as proposed in (A, c) may also increase strand transfer complexity.

Figure 6. hDNA and gene conversion tract length.

(A) Percentage of strand transfers associated with COs from wild type (black) and msh2Δ meioses (grey) as a function of tract length. ND corresponds to COs without detectable DNA strand transfer. (B) Percentage of strand transfers associated with NCOs from wild type (black) and msh2Δ meioses (grey) as a function of tract length. (C) Percentage of strand transfers associated with the three classes of msh2Δ NCOs as a function of tract length. Strand transfer patterns compatible with SDSA (black); trans hDNA patterns (dark grey); all other strand transfer patterns (light grey); all strand transfer patterns combined (white).

Figure 7. MMR–dependent conversion of transient hDNAs reconciles strand transfer patterns observed in the presence of Msh2 with those observed in the absence of Msh2.

The asterisks mark the hDNAs repaired by MMR. (A) Conversion of transient hDNA during canonical SDSA. On the left, the transient hDNA formed during the first 3′ end invasion is not repaired by MMR. Conversion of the second 3′ end only produces a short patch of conversion, while its restoration produces a silent event. On the right, the invading 3′ end is converted during strand invasion. Depending on the conversion or restoration of the second 3′ end, a long or a short patch of conversion is formed. In both cases the event is detectable. (B) During dHJ dissolution and double SDSA, conversion of the invading 3′ end combined with conversion of the second 3′ end after its capture can form a uniform conversion tract as observed for most of the NCOs in the presence of MMR. In this context, repair of a nick formed before or during resolution of the junction leads to a non-detectable event (b). (C) Conversion of transient hDNA during CO formation pathways I and II. Conversion of the invading 3′ end combined with conversion of the second 3′ end after its capture can form either a uniform 3∶1 conversion tract associated or not with a 2∶2 segregation tract (pathway (I)) or uniform 3∶1 conversion tract associated or not with a 1∶3 segregation tract (pathway (II)).