Abstract
Meiotic recombination is essential for producing healthy gametes, and also generates genetic diversity. DNA double-strand break (DSB) formation is the initiating step of meiotic recombination, producing, among other outcomes, crossovers between homologous chromosomes, which provide physical links to guide accurate chromosome segregation. The parameters influencing DSB position and repair are thus crucial determinants of reproductive success and genetic diversity. Using Schizosaccharomyces pombe, we show that the distance between sequence polymorphisms across homologous chromosomes has a strong impact on recombination, not only locally as intragenic events, but also on crossover frequency. This effect is controlled by MutS-MutL factors and DNA helicases. Additionally, we establish temperature as a major factor modulating meiotic recombination frequency, and identify DSB processing as a temperature-sensitive step in the meiotic recombination pathway. This exposes a complex interplay of genetic and environmental parameters shaping the outcome of meiotic recombination.
Introduction
Correct chromosome segregation during meiosis depends on pairing and physical connection of homologous chromosomes (homologs). Physical connections are established by the repair of programmed DNA double-strand breaks (DSBs) using the homolog rather than the sister chromatid as a template (i.e. interhomolog recombination) and by ensuring that interhomolog recombination intermediates are processed into crossovers (COs). The formation of DSBs by the transesterase Spo11 is thus a key step in initiating recombination during meiosis (Lam and Keeney 2015). Regions of high-frequency Spo11 recruitment, and thus DSB formation, are called hotspots (Wahls and Davidson 2012). One of the best characterized category of hotspots are cAMP-responsive elements, such as the ade6-M26 hotspot and its derivatives in Schizosaccharomyces pombe, created by point mutations in the ade6 gene (Wahls and Davidson 2012). M26-like hotspots are defined by the DNA sequence heptamer 5’-ATGACGT-3’, which represents the core of a binding site for the Atf1-Pcr1 transcription factor (Kon et al. 1997). Although binding of Atf1-Pcr1 and associated transcription already creates open chromatin at M26-like hotspots (Kon et al. 1997; Yamada et al. 2017), a very high frequency of meiotic recombination requires a conducive chromatin environment in a wider genomic context (Steiner and Smith 2005; Yamada, Ohta, and Yamada 2013). This network of parameters determines the overall level of DSB formation at a given genomic locus.
Following break formation, DSB ends are resected to initiate homologous recombination, which during meiosis follows either a Holliday junction/D-loop resolution or a synthesis-dependent strand annealing pathway (Lam and Keeney 2015; Hunter 2015). As a repair template, the sister chromatid or the homolog will be used (Humphryes and Hochwagen 2014). Based on this, it has been suggested that the governance of meiotic recombination could be viewed as a two-tiered decision system (Lorenz 2017). The first decision being template choice (interhomolog vs. intersister recombination), and the second being how the recombination intermediate is resolved - i.e. the CO/non-crossover (NCO) decision. The template choice decision is mainly driven by meiosis-specific factors of the chromosome axis and by the meiotic recombinase Dmc1 supported by its mediators (Humphryes and Hochwagen 2014). In budding yeast there is a basic understanding of how the interhomolog bias is established, although some mechanistic details still remain to be elucidated (Hong et al. 2013). Since homologs are not necessarily identical on a DNA sequence level, a DSB end invading the homolog for repair can generate a mismatch-containing heteroduplex DNA. Mismatches can be corrected by the mismatch repair system, consisting of the highly conserved MutS and MutL proteins (Surtees, Argueso, and Alani 2004). Additionally, the MutS-MutL complex can also block strand invasion to avoid recombination between non-homologous sequences (Surtees, Argueso, and Alani 2004). The CO/NCO-decision happens as the next step; here the decision is taken whether an already established interhomolog recombination intermediate is processed into a CO or a NCO. Determinants of the CO/NCO-decision are less well studied, but the DNA helicase/translocase FANCM (Fml1 in Sz. pombe) has been shown to limit CO formation in fission yeast and Arabidopsis (Lorenz et al. 2012; Crismani et al. 2012). RecQ-type DNA helicases perform a wide range of regulatory roles in homologous recombination, and one of them probably is the promotion of NCO formation during meiosis in various organisms (De Muyt et al. 2012; Lukaszewicz, Howard-Till, and Loidl 2013; Hatkevich et al. 2017).
In addition to these intrinsic genetic determinants, environmental factors play a role in dictating the outcome and dynamics of meiotic recombination. Environmental temperature has been identified as a modulating factor of meiotic recombination frequency in organisms incapable of regulating their body temperature (Bomblies, Higgins, and Yant 2015). The laboratory model yeasts Sz. pombe and Saccharomyces cerevisiae are globally distributed species with a poorly understood ecology (Liti 2015; Jeffares 2018), but it is likely that they are exposed to changing temperatures in their respective niches. Although a few observations about environmental temperature altering meiotic recombination have been made in the past in a variety of organisms, including yeasts (Plough 1917; Rose and Baillie 1979; Börner, Kleckner, and Hunter 2004; Pryce et al. 2005; Higgins et al. 2012), only recently more systematic approaches have explored the effect of the full temperature range at which meiosis is possible on meiotic recombination in a particular organism (Zhang et al. 2017; Lloyd et al. 2018; Modliszewski et al. 2018).
Here, we employ a series of genetic recombination assays featuring intragenic markers at differently sized intragenic intervals and flanking intergenic markers to identify and characterize intrinsic determinants of template choice and CO/NCO-decision in fission yeast. We show that the relative positions of DNA sequence polymorhisms between homologs have a strong impact on recombination outcome, not only locally in the form of intragenic recombination, but also on the CO frequency between an up- and a downstream marker. The anti-recombinogenic activity of MutS-MutL factors, and of the DNA helicases Fml1 and Rqh1 modulate recombination outcome differentially when comparing various intragenic intervals. Furthermore, we provide evidence how a simple environmental factor, such as temperature, influences recombination outcome locally, and identify DSB processing as the likely temperature-sensitive step of meiotic recombination.
Results
Rationale of the meiotic recombination assay
Our genetic recombination assay features intragenic markers (point mutations in the ade6 gene) and flanking intergenic markers (his3+-aim and ura4+-aim2) (Figure 1A-B). This assay allows us to monitor various recombination outcomes: (I) intragenic recombination events producing ade6+ recombinants, (II) crossovers (COs) between the flanking intergenic markers (his3+-aim and ura4+-aim2), and (III) the ratio of COs vs. non-crossovers (NCOs) among intragenic ade6+ recombination events (Figure 1A). Changes in intragenic recombination and overall CO frequencies observed in this assay can be explained by an altered frequency of DSB formation at a given ade6 mutant allele, or a change in repair template usage. The percentage of COs and NCOs among intragenic ade6+ recombination events is the genetic readout for the CO/NCO-decision, representing recombination intermediate processing after successful strand exchange between homologs. These events can be the result of gene conversions associated with COs or NCOs (non-reciprocal exchange of hereditary information), or of intragenic COs as a result of recombination intermediate resolution between the two point mutations within ade6 (reciprocal event) (Figure 1A, Figure 1-figure supplement 1; see below for details).
The physical distance between point mutations of heteroalleles defines the frequency of intragenic recombination events and their associated CO/NCO ratio
Apart from absolute DSB levels, intragenic recombination frequency is also influenced by the distance between point mutations in a given chromosomal region (Gutz 1971; Zahn-Zabal and Kohli 1996; Fox et al. 1997; Steiner and Smith 2005). Intragenic recombination in our assays (Figure 1A) has so far been monitored using point mutations within the ade6 coding sequence, which are at least 1kb apart (Osman et al. 2003; Lorenz, West, and Whitby 2010; Lorenz et al. 2012). We wondered whether the level of COs among intragenic recombination events also changes, when the distance between point mutations was decreased. Therefore, we selected a series of point mutations, which cover almost the complete length of the ade6 coding sequence (Figure 1B, Figure 1-table supplement 2). These point mutants include the strong meiotic recombination hotspots ade6-M26, - 3074, -3083, at the 5’ end of the gene and -3049 at the 3’ prime end of the gene, as well as the weak hotspot ade6-M375, and the non-hotspot alleles ade6-M216, -704, -52, -149, -51, and -469 (Figure 1B, Figure 1-table supplement 2). All strong hotspots mimic a cAMP-response element/Atf1-Pcr1 binding site (Kon et al. 1997; Steiner and Smith 2005). It can be safely assumed that a given hotspot will receive the same amount of breakage independent of the ade6 allele present on the homolog. This means that the differences seen in the combinations of one specific hotspot with various ade6 alleles will depend on processes downstream of DSB formation. Indeed, the frequency of intragenic recombination positively correlates with the distance between the ade6 alleles, when the same hotspot is used (Figure 1C, black and grey lines). The weak hotspot allele ade6-M375, which is at a similar position as the strong hotspot alleles ade6-3074 & ade6-3083, induces recombination only moderately. However, intragenic recombination frequency at ade6-M375 shows a similar correlation with respect to distance between the DNA polymorphisms (Figure 1C, green line). Intragenic intervals of similar size containing the meiotic recombination hotspot alleles, ade6-3083, ade6-3074, or ade6-3049, and a non-hotspot allele produce equivalent intragenic recombination levels (Figure 1C). Therefore, these hotspot alleles behave similarly in determining intragenic recombination frequency. Intriguingly, these observations are also largely true for CO frequency among intragenic recombination events. The shorter an intragenic distance between polymorphisms is, the more likely an intragenic recombination event is resolved as a NCO (Figure 1D). For crosses involving the hotspot alleles ade6-3083 or ade6-3074 the effect apparently tails off at intragenic distances >600bp (Figure 1D). Combining hotspot alleles on both homologs within a cross results in increased overall intragenic recombination rate compared with hotspot × non-hotspot crosses (Figure 1E), similar to what was previously reported (Hyppa and Smith 2010). However, there is no notable increase in COs among intragenic recombination events when compared to hotspot × non-hotspot crosses with similar intragenic distance between point mutations (Figure 1F). This indicates that the frequency of CO among intragenic recombination events is a function of the distance between the ade6 heteroalleles on the homologs. The distribution of different NCO/CO classes amongst intragenic recombination events follows a pattern consistent with intragenic NCOs events more likely being associated with the hotter allele. This means that the allele more likely to receive a DSB is the recipient of genetic information in the overwhelming majority of cases, which might represent a bona fide gene conversion (Figure 1G). If comparable hotspots are combined in a cross the two intragenic NCO classes occur with roughly equal frequency (Figure 1G, compare cross ade6-3083× ade6-3049 to crosses ade6-3083×ade6-469 & ade6-M375×ade6-3049).
The observed distribution patterns also suggest that, at these long intragenic intervals, a subset of CO events could stem from the processing of one joint molecule, presumably a single Holliday junction (Cromie et al. 2006) or its precursors, positioned between the two ade6 point mutations, in contrast to a gene conversion event being resolved as a CO. This hypothesis makes the following prediction: If CO events among Ade+ recombinants (mostly Ura- His- genotypes) are created by processing of a joint molecule situated between the two ade6 point mutations, then reciprocal Ade-Ura+ His+ recombinants carrying the mutations of both ade6 heteroalleles must exist. To test this, we sequenced the ade6 locus from 32 Ade-Ura+ His+ colonies from an ade6-3083×ade6-469 cross. Based on the frequency of 0.677% Ade+ Ura- His- events among the total viable progeny in such a cross representing 8.375% of recombinants among all Ura- His- colonies (240 Ura- His- colonies among 2,969 total viable progeny, 8.083%), we would expect that 2-3 of the 32 Ade- Ura+ His+ carry both the 3083 and the 469 mutation within the ade6 locus, if all events were generated by CO processing of a recombination event between the two heteroalleles. Indeed, we observed 2 instances in which the ade6 locus of Ade- Ura+ His+ progeny harbored both mutations (Figure 1-figure supplement 1), supporting the existence of intragenic COs (Figure 1A).
MutSα and MutLα are strong negative modulators of recombination frequency specifically at short intragenic intervals
Potential candidates for genetic pathways modulating recombination frequency at intragenic intervals of different lengths are MutS-MutL complexes which bind to heteroduplex DNA and repair mismatches (Surtees, Argueso, and Alani 2004). Sz. pombe has a streamlined nuclear mismatch repair system consisting of MutSα (Msh2-Msh6), MutSβ (Msh2-Msh3), and a single MutL (MutLα, Mhl1-Pms1); there is also a mitochondrial MutS protein called Msh1 (Marti, Kunz, and Fleck 2002). Importantly, the meiotic pro-crossover factors MutSγ (Msh4-Msh5), the meiosis-specific MutLγ component Mlh3, and Mlh2 – a MutLβ-homolog and a modulator of meiotic gene conversion tract length – are all missing in fission yeast (Manhart and Alani 2016; Duroc et al. 2017). This suggests that Sz. pombe is a suitable model to study the role of MutSα/β-MutLα during meiosis without potential crosstalk from MutSγ-MutLγ pro-crossover factors (Rogacheva et al. 2014).
At small intragenic intervals the absence of MutSα-MutLα causes a substantial increase in intragenic recombination frequency (Figure 2A, Figure 2-figure supplement 1). This relationship shows an inverse correlation, i.e. the shorter the intragenic interval the higher the increase. This ranges from a ~70-fold increase at the ade6-149×ade6-3049 (33bp) interval, via a ~35-fold one at ade6-3049×ade6-51 (53bp), to a ~10-fold augmentation at the ade6-M216×ade6-3083 (85bp) interval (Figure 2A, Figure 2-figure supplement 1). The MutSα mutants (msh2-30, msh6Δ) and the MutLα mutants (mlh1Δ, pms1-16) displayed similar frequencies of intragenic recombination to each other, and the msh2-30 mlh1Δ double mutant is not discernible from either single mutant (Figure 2A), indicating that MutSα and MutLα work in the same pathway. Deleting MutSβ (msh3) is of no consequence at the ade6-M216×ade6-3083 interval (Figure 2A; p=0.613 against wild type, two-tailed Mann-Whitney U), likely because all the ade6 mutations tested are substitution mutations, and MutSβ only recognizes insertion/deletion loop mismatches larger than 2 nucleotides (Surtees, Argueso, and Alani 2004). At larger intragenic intervals, there seems to be little or no role for MutSα-MutLα in limiting recombination events. In fact, a moderate, but mostly non-significant, tendency of lower intragenic recombination frequency can be observed (Figure 2B, Figure 2-figure supplement 1). Altogether, these data show that MutSα-MutLα has a strong anti-recombinogenic role at small intragenic intervals, but seemingly no role in determining recombination outcome at large intragenic intervals.
Mutating mutSα-mutLα genes increases CO frequency among intragenic recombination events (Figure 2C-D, Figure 2-figure supplement 2) and/or changes the distribution of recombinant classes (Figure 2-figure supplement 3). Both long and short intragenic intervals involving the ade6-3083 allele showed increases in associated CO frequency in comparison to wild type, albeit this trend was not statistically significant in all cases (Figure 2C-D, Figure 2-figure supplement 2). This trend makes the share of COs among intragenic recombination events independent of the length of the interval (compare Figure 1D with Figure 2C-D, Figure 2-figure supplement 2). Interestingly, there is also a substantial shift in CO classes among intragenic recombination events from mostly ura- his- to mainly ura+ his+ in mutSα-mutLα mutants at the short ade6-M216×ade6-3083 interval (Figure 2-figure supplement 3). This is not a consequence of selective survival or the formation of diploid or disomic spores, because mutSα-mutLα mutants have a spore viability similar to wild type, and the extent of the phenotype is the same in several different mutants (Supplementary File 1-Table S2). The possible significance of this finding is considered in the Discussion. As with intragenic recombination frequency, the mutSβ deletion msh3Δ behaves just like wild type for CO outcome (Figure 2C-D; p=0.439 against wild type, two-tailed Mann-Whitney U).
Fml1 is a negative modulator of intragenic CO frequency independent of the distance between point mutations
The DNA helicases, Fml1 and Rqh1, are also prime candidates for modulating recombination frequency at intragenic intervals of different lengths (Lorenz et al. 2012; Cromie, Hyppa, and Smith 2008). However, Fml1 apparently does not modulate intragenic recombination levels, as at all intragenic intervals tested, fml1Δ is similar to wild type (Figure 3A-B, Figure 3-figure supplement 1A). In contrast, the RecQ-family DNA helicase Rqh1 is required for wild-type levels of intragenic recombination (Lorenz et al. 2012). The deletion of rqh1 reduces intragenic recombination frequency to about a third of wild-type percentage at short (ade6-M216×ade6-3083, ade6-3049×ade6-469) intervals, and to about a tenth of wild-type frequency at the long ade6-3083×ade6-469 interval (Figure 3A-B, Figure 3-figure supplement 1).
As with long intervals (Lorenz et al. 2012) fml1Δ results in a ~10 percentage point increase of CO frequency among intragenic recombination events at short intervals (Figure 3C-D, Figure 3-figure supplement 1). The absence of Rqh1 induces moderate increases in CO levels among intragenic recombination events at the 85bp ade6-M216×ade6-3083 and the 1,3520bp ade6-3083×ade6-469 interval, which are not statistically significant (Figure 3C-D). However, at the 254bp ade6-3049×ade6-469 interval CO frequency among ade6+ events is raised by 17 percentage points in rqh1Δ (p=3.72×10-9 against wild type, two-tailed Mann-Whitney U) (Figure 3-figure supplement 1). Because ade6-3083 is a more complex allele than ade6-3049 (see Discussion), this potentially indicates that Fml1 can drive NCO pathway(s) independently of the complexity of the underlying DNA sequence, whereas Rqh1 can fulfill this role only at simple ade6 alleles with a single substitution mutation. Overall, these data show that Fml1 has no role in modulating intragenic recombination levels, but drives NCO formation downstream after successful strand invasion and DNA synthesis. Rqh1 promotes intragenic recombination, but also has moderate anti-recombinogenic activity in CO formation among intragenic recombination events.
The “fertile range” of fission yeast lies between 11°C and 33°C
Bomblies and coworkers recently noted that to understand the extent to which temperature affects meiotic recombination, it is important to know the “fertile range” of the tested species; otherwise the results will be skewed by including temperatures outside the “fertile range” or omitting temperatures within it (Lloyd et al. 2018). We set up matings of prototrophic fission yeast strains (ALP714×ALP688) in a temperature range between +4°C and +35°C on sporulation media. Matings were checked regularly until asci containing spores were observed, or, if not, the experiment was abandoned after 30 days. No asci were observed at +4°C and at +35°C after one month of incubation, putting the “fertile range” of Sz. pombe somewhere between these two temperatures. Indeed, mating at 11°C resulted in the formation of asci containing spores within 2 weeks, at 16°C within 1 week, at 20°C within 5 days, at 25°C and 30°C within 3 days, and at 33°C within 2 days. Sporulation efficiency was calculated as the percentage of asci containing spores in a given population of cells after the indicated time at each temperature. Sporulation efficiency was ~25% at all temperatures, except at 11°C when it was only ~5% (Figure 4A).
During the following meiotic recombination assays performed at 11°C, 16°C, 20°C, 25°C, 30°C and 33°C (“fertile range”), we also monitored spore viability by random spore analysis. At all temperatures tested, spore viability was ~60% (Figure 4B), indicating that at 11°C when sporulation is comparably inefficient (Figure 4A), the spores that developed did not suffer from decreased viability.
Meiotic intragenic recombination levels vary greatly within the “fertile range”
To assess whether temperature alters meiotic recombination outcome, assays were performed at temperatures within the “fertile range”. We tested five different combinations of ade6 heteroalleles: two large intragenic intervals containing a strong hotspot allele (ade6-3083×ade6-469, 1,320bp & ade6-M216×ade6-3049, 1,168bp), one large intragenic interval containing a weak hotspot allele (ade6-M375×ade6-469, 1,335bp), and two small intragenic intervals containing a strong hotspot allele (ade6-M216×ade6-3083, 85bp & ade6-3049×ade6-469, 254bp). The frequency of intragenic recombination is considerably lower at colder temperatures (11°C, 16°C and 20°C), and tends to plateau between 25°C and 33°C (Figure 4C-E). One of the large intervals (ade6-3083×ade6-469) displayed a distinct peak at 30°C (p=2.67×10-11 25°C vs. 30°C, p=2.6×10-5 30°C vs. 33°C; two-tailed Mann-Whitney U test). Intriguingly, the fold-change in intragenic recombination frequency between 16°C (lowest temperature tested in all intervals) and the temperature producing the highest intragenic recombination frequency is substantially lower in the cross with the weak ade6-M375 hotspot (2.7-fold) than in the crosses containing a strong hotspot allele (5-fold to 11-fold) (Supplementary File 1-Table S4). This also holds true if intragenic recombination frequency is compared between 16°C and 25°C (the mating temperature generally used for this type of experiment): 2.4-fold change in ade6-M375×ade6-469 vs. a 4.3- to 6.6-fold change in the crosses containing a strong hotspot allele (Supplementary File 1-Table S4). The very short ade6-M216×ade6-3083 intragenic interval (85bp) shows a stronger fold-change over temperature (6.6- fold at 16°C vs. 25°C), than the longer intervals containing a hotspot allele (254bp – 1,320bp; 4.3- to 4.8-fold at 16°C vs. 25°C) (Supplementary File 1-Table S4). This suggests, (I) that, as a general trend, lower temperatures reduce the frequency of intragenic recombination regardless of physical distance between ade6 mutations, (II) that intragenic recombination at weak hotspots is less sensitive to temperature changes than intragenic recombination at strong hotspots, and (III) that intragenic recombination at very short intervals is singularly susceptible to temperature changes.
Meiotic CO frequency varies moderately within the “fertile range”
Given that major changes in intragenic recombination levels are observed across temperatures, we were surprised to find that overall CO levels and CO frequencies among intragenic events were less sensitive to temperature changes. The frequency of COs between ura4+-aim2 and his3+-aim are not substantially altered as crossing temperature changes (Figure 4-figure supplement 1). In all intervals tested CO frequency in the total population is only significantly lower at the temperatures of 11°C and 16°C, but then plateaus at 20°C and higher (Figure 4-figure supplement 1A-C; Tukey’s Honest Significant Differences). CO frequency among intragenic ade6+ events was even more stable with temperature changes. The weak hotspot cross ade6-M375×ade6-469 was completely unfazed by temperature changes (p=0.314, Kruskal-Wallis test). The crosses at cold temperatures (11°C, 16°C & 20°C) in all the other intervals displayed a moderate tendency to higher CO percentages than crosses at 30°C or 33°C (Figure 4-figure supplement 1; Tukey’s Honest Significant Differences). The latter observation could indicate a mechanism like CO homeostasis at work (Martini et al. 2006; Kan, Davidson, and Wahls 2011).
Meiotic DSB levels do not appear to change with temperature
Following the observation that temperature modulates meiotic recombination outcome, we next sought to pinpoint which specific steps during meiotic recombination are sensitive to temperature changes. Therefore, we assessed whether DSB formation is likely disturbed using the cytological markers Rec7-GFP and Rad11-GFP. Rec7 (Rec114 in S. cerevisiae), one of the co-factors essential for Spo11 recruitment and function (Miyoshi, Ito, and Ohta 2013), can be detected on meiotic chromatin and is considered a marker for DSB initiation sites (Lorenz et al. 2006). As part of RPA (replication protein A) Rad11 becomes associated with the single-stranded DNA exposed by strand resection following removal of Spo11, and is thus a marker for DSB formation (Parker et al. 1997). Rec7- and Rad11-focus numbers enable us to assess meiotic DSB levels indirectly. For Rec7- and Rad11-focus counts, linear elements outlined by myc-tagged Hop1 were used to identify meiotic prophase I nuclei in chromatin spreads from meiotic time-courses (Lorenz et al. 2004; Brown, Jarosinska, and Lorenz 2018; Loidl and Lorenz 2009). We chose to perform these experiments at the extreme temperatures of the “fertile range” (16°C and 33°C), which were still producing high sporulation efficiency and significantly different recombination frequencies (Figure 4).
Based on previous observations that recombination markers are most abundant in the thread and network stage of linear element formation (Lorenz et al. 2006), we selectively counted foci at these stages. On average between ~16 foci of both Rec7-GFP and Rad11-GFP per nucleus were observed at 16°C and 33°C (Figure 5A-B). The Rec7-GFP focus count was actually somewhat higher at the lower temperature (18.2 at 16°C vs. 14.1 at 33°C, p=0.0017, two-tailed Mann-Whitney U test), whereas the Rad11-GFP focus numbers were indiscernible between 16°C (15.9 foci/nucleus) and 33°C (16.2 foci/nucleus) (Supplementary File 1-Table S5; p=0.794, two-tailed Mann-Whitney U test).
These experiments suggest that overall DSB formation is largely unaltered between 16°C and 33°C, because any subtle changes observed are unlikely to explain the lowered recombination frequencies at cold temperatures.
Processing of DSBs is potentially altered by temperature
Rqh1 and Exo1 function in long-range strand resection in mitotic and meiotic cells in fission yeast (Langerak et al. 2011; Osman et al. 2016). Sfr1 forms a complex with Swi5 to support strand exchange, thereby promoting meiotic recombination (Haruta et al. 2006; Lorenz et al. 2012). Less efficient DSB processing and/or strand exchange could potentially explain why recombination levels are reduced at colder temperatures. The expectation would be that mutants defective in strand resection or strand exchange would be more sensitive to temperature changes than wild type (i.e., a synergistic effect of mutational and environmental weakening of these processes). Therefore, meiotic recombination outcome in ade6-3083×ade6-469 crosses of rqh1Δ, exo1Δ, and sfr1Δ single mutants performed at 16°C and 25°C was determined. The fold difference in intragenic recombination frequency between 16°C and 25°C for wild type and each deletion was calculated to assess whether the reduction in intragenic recombination at cold temperatures is epistatic or synergistic with deleting rqh1, exo1, or sfr1. In wild-type crosses intragenic recombination is 4.3-fold lower at 16°C compared to 25°C (p=6.428×10-12, two-tailed Mann-Whitney U test). However, in rqh1Δ, exo1Δ, and sfr1Δ crosses intragenic recombination levels are 7.2-fold (p=1.402×10-9, two-tailed Mann-Whitney U test), 7.1-fold (p=4.665×10-11, two-tailed Mann-Whitney U test), and 7.9-fold (p=6.265×10-7, two-tailed Mann-Whitney U test) lower at 16°C than at 25°C, respectively (Figure 6). The fold changes in overall CO frequency and CO levels among ade6+ recombinants are largely unchanged or do not follow an obvious pattern (Supplementary File 1-Table S4). Long-range strand resection and the action of strand exchange factors are potentially important for maintaining intragenic recombination frequency especially at colder temperatures, indicating that these processes possibly are temperature-sensitive.
Discussion
Differences in the DNA sequences of the homologs affect recombination
We used a genetic recombination assay with ade6 as central marker gene to determine whether different distances between polymorphisms (intragenic interval) influence intragenic and intergenic recombination outcome (Figure 1). This potentially has implications for how we think about meiotic recombination. Rather than simple gene conversions at single loci, which are thought to primarily arise from mismatch repair or from DNA synthesis during DSB repair (Holliday 2007), intragenic recombination events involving two distinct point mutations on the homologs have the additional possibility of being caused by intragenic COs (Figure 1-figure supplement 1). This would imply that the occurrence of a CO between two point mutations is more likely the longer the distance between the two heteroalleles is, and that this will result in an intragenic event with a higher probability. This, admittedly, exposes a rather blurred boundary between what constitutes a bifactorial GC event associated with a CO and what an intragenic CO event. The three mechanisms of GC formation (mismatch repair, DNA synthesis during DSB repair, and intragenic COs) are not mutually exclusive, but to a degree even presuppose each other.
The observed effects for different parental and recombinant classes amongst progeny having undergone a meiotic intragenic recombination event can be explained by envisioning a DSB 5’ or 3’ of a point mutation leading to a recombination intermediate (D-loop, Holliday junction), which will then be processed immediately at the break site, or ends up somewhat removed from the initial break site by multiple consecutive invasion steps, by branch migration, or both (Farah, Cromie, and Smith 2009; Piazza, Wright, and Heyer 2017; Marsolier-Kergoat et al. 2018). The genetic makeup of the progeny is, therefore, a compound result of processing distinct recombination intermediates in different ways. The genetic composition of wild-type and mutant progeny resulting from the meiotic recombination assays can be explained as different combinations of scenarios suggested previously (Lorenz et al. 2014). For example, recombination between ade6-3083 and ade6-M216, which gives rise to mainly Ade+ His+ Ura- NCOs and Ade+ His- Ura- COs, may be explained by the model in Figure 7A. In this model, a bias in favour of Ade+ His- Ura- COs stems from strand exchange/branch migration being constrained to within the region defined by the ade6-3083 – ade6-M216 interval and resolution of the recombination intermediate occurring by D-loop cleavage (Figure 7A, C). Ade+ His+ Ura- NCOs and additional Ade+ His- Ura- COs come from HJ resolution (Figure 7A, C). However, certain mutant situations can dramatically alter the outcome, e.g. recombination at ade6-M216×ade6-3083 in mutSα-mutLα mutants leads to relatively few Ade+ His- Ura- COs and a big increase in the proportion of Ade+ His+ Ura+ COs (Figure 2, Figure 2-figure supplement 3). We considered whether this might have something to do with the complexity of the ade6-3083 allele, which consists of multiple substitution mutations and can potentially form a C/C-mismatch in the heteroduplex DNA during strand exchange that is less efficiently repaired during meiosis than other mismatches (Schär and Kohli 1993). However, a moderate shift of CO recombinant classes among intragenic events can also be seen at the small ade6-149×ade6-3049 interval (Figure 2-figure supplement 3). Unlike ade6-3083, ade6-3049 contains only a single nucleotide difference (Figure 1-table supplement 2) and, therefore, the complexity of a given ade6 allele is unlikely to be the critical factor affecting the shift in CO recombinant class. Instead, we think that a deficit in heteroduplex rejection and mismatch repair, caused by loss of msh2, could result in strand exchange/branch migration extending beyond the non-hotspot mutation (i.e. ade6-M216 or ade6-149) prior to D-loop cleavage/HJ resolution, and the base-pair mismatches in the recombinant chromosomes remaining unrepaired. Together, these altered features could explain the increase in Ade+ His+ Ura+ COs at the ade6-M216×ade6-3083 and ade6-149×ade6-3049 intervals in mutSα-mutLα mutant crosses (Figure 7B, C).
Recombination outcome in a msh2Δ in S. cerevisiae has also been shown to be more complex than in wild type (Martini et al. 2011; Cooper et al. 2018). Intriguingly, in S. cerevisiae the action of Msh2 seems to be restricted to class I COs, which are subjected to CO interference, whereas Mus81-dependent class II COs are unchanged in msh2Δ (Cooper et al. 2018). Sz. pombe operates only a class II CO pathway via Mus81-processing, completely lacking a class I CO pathway. Nevertheless, the absence of Msh2 in fission yeast has a profound effect on CO frequency, and the way recombination intermediates are processed (Figure 2).
FANCM- and RecQ-family DNA helicases/translocases are implicated in regulating meiotic recombination outcome in several different organisms (Lorenz et al. 2012; Crismani et al. 2012; De Muyt et al. 2012; Cromie, Hyppa, and Smith 2008; Hatkevich et al. 2017; Lukaszewicz, Howard-Till, and Loidl 2013). In Sz. pombe Fml1 has been shown to specifically limit CO formation during the late CO/NCO-decision (Lorenz et al. 2012). Fml1 acts as a promotor of NCOs, likely by driving late recombination intermediates into the SDSA pathway, after strand invasion and DNA synthesis has happened. In accordance with this, absence of fml1 leads to an increase in CO among intragenic ade6+ events, but has little effect on intragenic recombination itself (Figure 3, Figure 3-figure supplement 1) (Lorenz et al. 2012). This role is independent of the size of the intragenic interval, with Fml1 driving 10-12% of NCO recombination in any case. The deletion of rqh1 has a very strong meiotic phenotype, leading to reductions in intragenic recombination, CO, and spore viability (Figure 3, Figure 3-figure supplement 1). This on its own would indicate an early role in promoting strand exchange and/or DSB resection, but then Rqh1 is capable of promoting NCO formation among ade6+ events at some intragenic intervals (Figure 3, Figure 3-figure supplement 1). Most likely this is due to Rqh1 actually performing the following functions: (I) promotion of interhomolog recombination events, probably in cooperation with Rad55-57 and Rlp1-Rdl1-Sws1, but independently of Sfr1-Swi5 (Lorenz et al. 2014), potentially also by providing longer resection tracts (Osman et al. 2016); (II) dismantling D-loops, this enables the release of break ends to search for homology elsewhere, starts cycles of multiple consecutive invasion steps, and provides opportunities for Fml1 to drive NCO formation via SDSA; and (III) branch migration of established D-loops and Holliday junctions, thereby promoting heteroduplex DNA formation further away from the break site (Cromie, Hyppa, and Smith 2008).
Environmental temperature influences recombination outcome
The environmental temperature regime during crossing affects meiotic recombination outcome in fission yeast, while DSB levels appear to be maintained across temperatures in the “fertile range” (Figures 4, 5). Steps in the meiotic recombination pathway that are downstream of DSB formation, such as strand resection and/or strand exchange are likely impaired at colder temperatures (Figure 6). Especially, intragenic recombination frequency shows strong changes with temperature within the “fertile range”, whereas overall COs and COs among intragenic events are less affected (Figure 4, Figure 4-figure supplement 1). Recombination monitored at non-hotspot alleles only are less sensitive to temperature changes than those involving a hotspot (Supplementary File 1-Table S4); this could be a manifestation of CO invariance suggested to explain a stronger drive towards interhomolog recombination at non-hotspots (Hyppa and Smith 2010). CO changes over temperature do definitely not follow a U-shape curve like in Drosophila or Arabidopsis (Plough 1917; Lloyd et al. 2018), where CO recombination is highest at the more extreme temperatures within the “fertile range”. Similar to C. elegans (Rose and Baillie 1979) CO rates tend to be lower at low temperatures (Figure 4-figure supplement 1). In Hordeum vulgare (barley) and S. cerevisiae CO position, rather than overall frequency, changes with temperature (Higgins et al. 2012; Zhang et al. 2017). In S. cerevisiae this has largely been explained by differential activation of DSB hotspots at different temperatures (Zhang et al. 2017). However, in contrast to S. cerevisiae where the location of DSBs is maintained in only ~20% of cases between temperatures (14°C, 30°C, and 37°C) (Zhang et al. 2017), in Sz. pombe this is true for ~70% of DSB sites (Hyppa et al. 2014). It is thus unlikely that changes of recombination frequency due to differential hotspot activation over temperature is a major contributing factor in Sz. pombe. Considering that overall CO frequency is only moderately affected by temperature, whereas intragenic recombination rates change massively, a switch from interhomolog to intersister recombination will unlikely be a key contributing factor, since this would affect intergenic COs and intragenic recombination to an equal extent. Processes directly downstream of DSB formation, such as strand invasion and stabilisation of strand exchange, are temperature-sensitive (Figure 6), and are seemingly a major cause for low intragenic recombination frequency at low temperatures.
Concluding remarks
Factors directly involved in generating CO and NCO recombinants during meiosis have been identified and characterized in recent years (Osman et al. 2003; De Muyt et al. 2012; Lorenz et al. 2012; Lukaszewicz, Howard-Till, and Loidl 2013; Crismani et al. 2012), and several inroads have been made in understanding how template choice is regulated and executed during meiotic recombination in standard laboratory conditions (Hong et al. 2013; Lorenz et al. 2014). However, we still only have a basic understanding of how underlying DNA sequence polymorphisms and environmental parameters influence meiotic recombination outcomes. Here, we demonstrate that specific DNA sequence differences between the two homologs strongly impact on which outcome is achieved, and that this is largely driven by the action of the MutS-MutL complex. Other important determinants influencing meiotic recombination outcome are environmental factors, such as temperature. Temperature changes within the “fertile range” of a species grossly affects intra- and intergenic recombination levels in several species (Plough 1917; Rose and Baillie 1979; Bomblies, Higgins, and Yant 2015; Lloyd et al. 2018), likely by changing the positioning of the initial DSB (Higgins et al. 2012; Zhang et al. 2017) and/or dynamics of DSB repair (Modliszewski et al. 2018; this study). This highlights the importance of the interplay between intrinsic and environmental parameters in shaping the genetic diversity of a given population.
Material and methods
Bacterial and yeast strains and culture conditions
E. coli strains were grown on LB and SOC media – where appropriate containing 100 μg/ml Ampicillin (Sambrook and Russell 2000). Competent cells of E. coli strains NEB10®-beta (New England BioLabs Inc., Ipswich, MA, USA), and XL1-blue (Agilent Technologies, Santa Clara, CA, USA) were transformed following the protocols provided by the manufacturers. Schizosaccharomyces pombe strains used for this study are listed in Supplementary File 2. Yeast cells were cultured on yeast extract (YE), and on yeast nitrogen base glutamate (YNG) agar plates containing the required supplements (concentration 250 mg/l on YE, 75 mg/l on YNG). Crosses were performed on malt extract (ME) agar containing supplements at a final concentration of 50 mg/l (Sabatinos and Forsburg 2010).
Different ade6 hotspot and non-hotspot sequences (Figure 1-table supplement 2) were introduced by crossing the respective mutant ade6 strain with ade6+ strains carrying the ura4+ and his3+ artificially introduced markers (aim) (UoA95, UoA96, UoA97, UoA98) (Osman et al. 2003). The point mutations in the ade6 alleles were verified by Sanger DNA sequencing (Source BioScience, Nottingham, UK) (Figure 1-table supplement 2).
Using an established marker swap protocol (Sato, Dhut, and Toda 2005) the natMX6-marked rqh1Δ-G1 was derived from an existing rqh1Δ::kanMX6 allele (Doe et al. 2002), creation of the natMX6-marked pms1-16 insertion mutant allele has been described previously (Lorenz 2015).
Marker cassettes to delete msh3, and msh6, and to partially delete msh2 were constructed by cloning targeting sequences of these genes into pFA6a-kanMX6, pAG25 (natMX4), and pAG32 (hphMX4), respectively, up- and downstream of the dominant drug resistance marker (Bähler et al. 1998; Goldstein and McCusker 1999). The targeting cassettes were released from the relevant plasmids (pALo130, pALo132, pALo134) by a restriction digest, and transformed into the strains FO652 (msh2 and msh6) and ALP729 (msh3). For specifics of strain and plasmid construction please refer to Supplementary File 3. Plasmid sequences are available on figshare (https://figshare.com/s/ad72dbfe07a261fd4ee4). Epitope tagging of hop1+ with a C-terminal 13myc-kanMX6 tag has been described in detail (Brown, Jarosinska, and Lorenz 2018).
Transformation of yeast strains was performed using an established lithium-acetate procedure (Brown and Lorenz 2016). All plasmid constructs were verified by DNA sequencing (Source BioScience plc, Nottingham, UK).
All DNA modifying enzymes (high-fidelity DNA polymerase Q5, restriction endonucleases, T4 DNA ligase) were supplied by New England BioLabs. Oligonucleotides were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).
Genetic and cytological assays
Determination of spore viability by random spore analysis and the meiotic recombination assay have been previously described in detail (Osman et al. 2003; Sabatinos and Forsburg 2010).
Genomic DNA of Ade- Ura+ His+ progeny from an ade6-3083×ade6-469 (ALP733×ALP731) cross was used to PCR-amplify the ade6 locus (oligonucleotides oUA219 5’-AAAGTTGCATTTCACAATGC-3’ and oUA66 5’-GTCTATGGTCGCCTATGC-3’) for Sanger sequencing (Eurofins Scientific, Brussels, Belgium) with oUA219, oUA66, or nested oligonucleotides oUA779 5’-CTCATTAAGCTGAGCTGCC-3’ and oUA780 5’-AAGCTCTCCATAGCAGCC-3’.
Meiotic time-courses and preparation of chromatin spreads were in essence performed as described previously (Loidl and Lorenz 2009), except for the use of 100 mg/ml Lallzyme MMX (Lallemand Inc., Montréal, Canada) as the only cell-wall digesting enzyme in the spheroplasting solution of the chromatin spread protocol (Flor-Parra et al. 2014). Immunostaining was performed according to an established protocol (Loidl and Lorenz 2009) using polyclonal rabbit α-myc (ab9106; Abcam PLC, Cambridge, UK) at a 1:500 dilution and monoclonal rat α-GFP [3H9] (ChromoTek GmbH, Planegg-Martinsried, Germany) at a 1:100 dilution as primary antibodies. Antibody-bound protein was visualized using donkey α-rabbit IgG AlexaFluor-555 (ab150062; Abcam) and donkey α-rat IgG AlexaFluor-488 (ab150153; Abcam), both at a 1:500 dilution, as secondary antibodies conjugated to fluorophores. DNA was stained by Hoechst 33342 (Molecular Probes, Eugene, OR, USA) at a final concentration of 1 μg/ml.
Analysis was performed on a Zeiss Axio Imager.M2 (Carl Zeiss AG, Oberkochen, Germany) epifluorescence microscope equipped with the appropriate filter sets to detect red, green, and blue fluorescence. Black-and-white images were taken with a Zeiss AxioCam MRm CCD camera controlled by AxioVision 40 software v4.8.2.0. Images were pseudo-coloured and overlayed using Adobe Photoshop CC (Adobe Systems Inc., San José, CA, USA).
For Rec7-GFP and Rad11-GFP focus counts, images of meiotic prophase I nuclei, as identified by the presence of Hop1-13myc linear elements at the thread and network stages (Lorenz et al. 2006), were captured as described using the above antibodies. Individual images were acquired for each channel to detect Hop1-13myc, either Rec7-GFP or Rad11-GFP, and the DNA stain Hoechst 33342. Single image channels were merged, and all GFP-positive foci counted within the area defined by the Hoechst 33342 staining using the “count” function in Adobe Photoshop CC.
Data presentation and Statistics
Raw data is available on figshare (https://figshare.com/s/ad72dbfe07a261fd4ee4). Line graphs were produced using Microsoft Excel 2016 (version 16.0.4638.1000, 32-bit), and scatter plots were generated in GraphPad Prism 5 for Windows (version 5.04). Box-and-whisker plots were created in R (version i386, 3.0.1) (http://www.r-project.org/) (Lorenz et al. 2014). R was also used to compute Kruskal-Wallis test and Tukey’s Honest Significant Differences employing the kruskal.test() and TukeyHSD() functions, respectively. Mann-Whitney U tests were performed as previously described (Lorenz et al. 2014).
Author contributions
SDB: conception and design, unpublished essential reagents (yeast strains, plasmids), acquisition of data, analysis and interpretation of data, revising the manuscript; MNA, MJ, CA, SJM: acquisition of data, revising the manuscript; MCW: analysis and interpretation of data, revising the manuscript; AL: conception and design, unpublished essential reagents (yeast strains, plasmids), acquisition of data, analysis and interpretation of data, drafting and revising the manuscript.
Acknowledgments
We are grateful to Jürg Bähler, Miguel G. Ferreira, Edgar Hartsuiker, Franz Klein, Jürg Kohli, Josef Loidl, Kim Nasmyth, Fekret Osman, Gerald R. Smith, Walter W. Steiner, and the National BioResource Project (NBRP) Japan for providing materials, and to C. Bryer, A. Mehats, and H. Rickman for technical assistance. This work was supported by the Biotechnology and Biological Sciences Research Council UK (BBSRC) [grant numbers BB/F016964/1, BB/M010996/1], the University of Aberdeen (College of Life Sciences and Medicine Start-up grant to AL), and the Wellcome Trust (Programme grant to MCW) [grant number 090767/Z/09/Z].