The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance

Abstract

Background: The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids.

Methodology/principal findings: In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination) and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of 'second strand capture' when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent) are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.

Conclusions: This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout meiosis.

Figure 1. Mis-segregation events during meiosis.

During meiosis, crossing over ensures the accurate segregation of homologs at meiosis I. Sister chromatids separate at meiosis II. In yeast, all four meiotic products are recovered as viable spores (A). The absence of crossovers may lead to both homologs becoming pulled towards the same pole at meiosis I. Meiosis I non-disjunction leads to two disomic spores (B). The inability to resolve entangled chromosomes can lead to chromosome breakage. A centromere marker on a pair of normally segregating chromosomes can be used to identify the sister and non-sister spores. In the case of meiosis I non-disjunction, these are sister spores (B). In Figure 1C the inability to resolve the crossover leads to the two viable spores being non-sisters (C).

Figure 2. Structural and functional domains of Sgs1.

The interacting domains of SGS1 (shown with amino acid coordinates) highlighting the point mutations used in this study that disrupt the Top3-interacting domain of Sgs1 (sgs1-top3-id) and disrupt the Mlh1-interacting domain of Sgs1 (sgs1-mlh1-id). The RecQ Conserved (RQC) domain facilitates protein-protein interactions. The Helicase-and-RNaseD-C-terminal (HRDC) domain is required for DNA binding. The helicase domain facilitates the unwinding of recombination intermediates. Sgs1 also interacts with Top2 and the Top2-interacting domain of Sgs1 overlaps the helicase domain and two highly acidic regions (AR) found in the protein (as described in the Introduction ).

Figure 3. Map distances on chromosome III.

(A) Homologous recombination. B) Homeologous recombination. Map distances were calculated using the Perkins formula . The distribution of PDs, NPDs and TTs for homologous diploids was compared using the G-test. After correcting for multiple comparisons using the Benjamini-Hochberg correction , p-values <0.05 were considered significant. The standard error of the map distances was calculated using Stahl Online Tools (http://molbio.uoregon.edu/~fstahl/compare2.php). * - significantly different from WT/WT; # - significantly different from sgs1Δ/sgs1Δ; † - significantly different from sgs1-ΔC795/sgs1Δ; ± - significantly different from pCLB2-SGS1/sgs1Δ.

Figure 4. Frequency of meiosis I non-disjunction events for homeologous diploids.

Meiosis I non-disjunction events on chromosome III were identified in homeologous diploids as described in Methods and Materials . Frequencies of meiosis I non-disjunction events out of the total number of tetrads dissected were compared using the G-test. After correcting for multiple comparisons using the Benjamini-Hochberg correction , p-values <0.05 were considered significant. * - significantly different from WT/WT; # - significantly different from sgs1Δ/sgs1Δ.

Figure 5. Unequal Recombination.

A strain containing the HYG-CYH/HYG cassette (in red) was mated to a strain that does not (in green) to assess unequal recombination events. The genetic (drug resistance/sensitivity) phenotype and physical karyotype of all four spores from each type of recombination event are illustrated. A: A reciprocal crossover event that occurs between HIS4 and LEU2, leading to 3 Hyg^R: 1 Hyg^S and 2 Cyh^S: 2 Cyh^R. B: Intra-chromatid events (or Deletion events). Crossing over between the hygromycin cassettes on the same sister strand lead to a deletion event. This is seen as 2 Hyg^R: 2 Hyg^S and 1 Cyh^S: 3 Cyh^R segregation patterns. Four lanes of a CHEF Gel are shown, each of which represents one spore of a four viable spore tetrad. Chromosome III is indicated with an arrow (→). Due to the deletion event, approximately 27.5kb DNA will have been lost. This results in the absence of a band where expected and a band of double intensity below, as chromosome III now migrates with chromosome VI. When probed with URA3 and CYH2 sequences, the URA3 containing chromosome V (top band) and the CYH2 containing chromosome VII (middle band) are labelled. Chromosome III (bottom band) is labelled when it retains the CYH2 insert. Thus, the smaller chromosome III, which has deleted all of the sequences between HIS4 and LEU2, is unlabelled. C: Inter-chromatid events (or Unequal Sister Chromatid Exchange events). When a reciprocal crossover occurs between one hygromycin cassette on one sister strand and the other hygromycin cassette on the second sister strand, a triplication event and a deletion event are seen as 2 Hyg^R: 2 Hyg^S and 1 Cyh^S: 3 Cyh^R colonies. The triplication event results in chromosome III migrating more slowly, while the deletion event migrates faster (as discussed in B). Southern blot analysis is used as physical confirmation of the genetic diagnosis, as discussed above (B). D: Gene Conversion events. Tetrads that are 2 Hyg^R: 2 Hyg^S and 1 Cyh^S: 3 Cyh^R can also arise by gene conversion of the HYG-CYH region. Because a gene conversion event does not result in a major size change, the CHEF karyotype is normal. However, Southern blotting indicates that one copy of the CYH2 gene has been replaced with wild-type sequences.

Figure 6. Failure to decatenate sister chromatids can lead to spore inviability.

A: Sgs1 acts in the decatenation of dHJ structures with Top3 late during meiosis. Sgs1 and Top3 act to dissolve double Holliday junctions (i). In the absence of this interaction, the double Holliday junction is not dissolved leading to destruction of the entangled chromosomes and inviability of the non-sister spores (ii). B: Sgs1 interacts with Top2 to decatenate sister chromatids arising from pre-meiotic replication. Failure to decatenate sister chromatids (iii) lead to the inability of recombined chromosomes to segregate. The entanglement can lead to chromosome breakage and/or chromosome loss (iv). Because the crossover links the non-sister centromeres, the two remaining viable spores are also non-sisters.