Abstract
Meiotic chromosomes are divided into regions of enrichment and depletion for meiotic chromosome axis proteins, in budding yeast Hop1 and Red1. These proteins are important for formation of Spo11-catalyzed DSB, but their contribution to crossover recombination is undefined. By studying meiotic recombination initiated by the sequence-specific VMA1-derived endonuclease (VDE), we show that meiotic chromosome structure helps to determine the biochemical mechanism by which recombination intermediates are resolved to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers required the MutLγ resolvase, which forms most Spo11-initiated crossovers. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and also displayed similar MutLγ-dependence of VDE-induced crossovers. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, with features of meiotic chromosome structure partitioning the genome into regions where one pathway or the other predominates.
Introduction
The transition from the mitotic cell cycle to meiosis involves substantial changes in mechanisms of DNA double strand break (DSB) repair by homologous recombination (HR). Most mitotic HR repairs spontaneous lesions, and most repair products are non-crossovers (NCOs) that do not involve exchange of flanking parental sequences (Fabre et al., 1984; Kadyk and Hartwell, 1992) (Ira et al., 2003; Kelly, 1974; Pâques et al., 1998; Stark and Jasin, 2003; Taghian and Nickoloff, 1997; Virgin et al., 2001). In contrast, meiotic recombination is initiated by programmed DSBs (Cao et al., 1990; Sun et al., 1989) that often are repaired as crossovers (COs) between homologous chromosomes (homologs), with exchange of flanking parental sequences. Inter-homolog COs create physical linkages, called chiasmata, that ensure faithful homolog segregation during the first meiotic division, avoiding chromosome nondisjunction and consequent aneuploidy in gametes [reviewed by (Hunter, 2015)].
The DSBs that initiate meiotic recombination are formed by Spo11 in complex with a number of accessory proteins, and will be referred to here as Spo11-DSBs [reviewed by (Lam and Keeney, 2015)]. Spo11-DSBs and resulting recombination events are non-uniformly distributed in the genomes of organisms ranging from budding yeast to humans (Baudat Nicolas, 1997; Blitzblau etal., 2007; Buhler etal., 2007; Gerton etal., 2000; Pan et al., 2011) (Fowler et al., 2013) (Wijnker et al., 2013) (Hellsten et al., 2013) (Singhal etal., 2015) (Smagulova etal., 2011) (Pratto etal., 2014). In budding yeast, this nonuniform distribution of Spo11-DSBs is influenced by meiosis-specific proteins, Red1 and Hop1, which are components of the meiotic chromosome axis. The meiotic chromosome axis coordinates sister chromatids and forms the axial element of the synaptonemal complex, which holds homologs in tight juxtaposition (Hollingsworth et al., 1990; Page and Hawley, 2004; Smith and Roeder, 1997; Sym et al., 1993). Spo11-DSBs form frequently in large (ca 50-200 kb) “hot” domains that are also enriched for Red1 and Hop1, and these “hot” domains are interspersed with similarly-sized “cold” regions where Spo11-DSBs are infrequent and Red1/Hop1 occupancy levels are low (Baudat and Nicolas, 1997; Blat et al., 2002; Blitzblau et al., 2007; Buhler et al., 2007; Pan et al., 2011; Panizza et al., 2011). Normal Spo11-DSB formation requires recruitment of Spo11 and accessory proteins to the meiotic axis (Panizza et al., 2011; Prieler et al., 2005), and Red1/Hop1 are also central to mechanisms that direct Spo11-DSB repair towards use of the homolog as a recombination partner (Carballo etal., 2008; Niu etal., 2005; Schwacha and Kleckner, 1997). Other eukaryotes contain Hop1 analogs that share a domain, called the HORMA domain (Rosenberg and Corbett, 2015), and correlations between these meiotic axis proteins and DSB formation are observed in fission yeast, nematodes and in mammals (Fowler et al., 2013; Goodyer et al., 2008; Wojtasz et al., 2009). Thus, most meiotic interhomolog recombination occurs in the context of a specialized chromosome structure and requires components of that structure.
Meiotic recombination pathways diverge after DSB formation and homolog-directed strand invasion. In budding yeast, about half of events form NCOs via synthesis-dependent strand annealing, a mechanism that does not involve stable recombination intermediates (Allers and Lichten, 2001a; Martini et al., 2011; McMahill et al., 2007) and is suggested to be the predominant HR pathway in mitotic cells (Bzymek et al., 2010; McGill et al., 1989; Mitchel et al., 2013). Most of the remaining events are repaired by a meiosis-specific CO pathway, in which an ensemble of meiotic proteins, called the ZMM proteins, stabilize early recombination intermediates and promote their maturation into double Holliday junction joint molecules (JMs) (Allers and Lichten, 2001a; Börner etal., 2004; Lynn etal., 2007; Schwacha and Kleckner, 1994). These ZMM-stabilized JMs are subsequently resolved as COs (Sourirajan and Lichten, 2008) through the action of the MutLγ complex, which contains the Mlh1, Mlh3, and Exo1 proteins (Argueso etal., 2002; 2004; Khazanehdari and Borts, 2000; Wang et al., 1999; Zakharyevich et al., 2010; 2012). MutLγ does not appear to make significant contributions to mitotic COs (Ira et al., 2003; Welz-Voegele et al., 2002). A minority of events form ZMM-independent JMs that are resolved as both COs and NCOs by the structure-selective nucleases (SSNs) Mus81-Mms4, Yen1, and Slx1-Slx4, which are responsible for most JM resolution during mitosis (Argueso etal., 2004; de los Santos etal., 2003; De Muyt et al., 2012; Zakharyevich et al., 2012) (Ho et al., 2010; Muñoz-Galván et al., 2012) [reviewed by (Wyatt and West, 2014)]. A similar picture, with MutLγ forming most meiotic COs and SSNs playing a minor role, is observed in several other eukaryotes (Falque et al., 2009; Franklin et al., 2006; Hassold et al., 2009; Kochakpour and Moens, 2008; Lhuissier etal., 2007; Plug et al., 1998; Tease and Hultén, 2004) (Higgins et al., 2008; Holloway etal.,2008).
To better understand the factors that promote the unique biochemistry of CO formation during meiosis, in particular MutLγ-dependent JM resolution, we considered two different hypotheses. In the first, expression of meiosis-specific proteins and the presence of high levels of Spo11-DSBs results in nucleus-wide changes in recombination biochemistry, shifting the balance towards MutLγ-dependent resolution of JMs, wherever they might occur. In the second, local features of meiotic chromosome structure, in particular enrichment for meiosis-specific chromosome axis proteins, provides an in cis structural environment that favors MutLγ-dependent JM resolution. However, because Spo11-DSBs form preferentially in Red1/Hop1-enriched regions, and because these proteins are required for efficient Spo11-DSB formation and interhomolog repair, it is difficult to distinguish these two models by examining Spo11-initiated recombination alone.
To test these two hypotheses, we developed a system in which meiotic recombination is initiated by the sequence- and meiosis-specific VMA1 derived endonuclease, VDE (Gimble and Thorner, 1992; Nagai et al., 2003). VDE initiates meiotic recombination at similar levels wherever its recognition sequence (VRS) is inserted (Fukuda et al., 2008; Neale et al., 2002; Nogami et al., 2002). VDE-catalyzed DSBs (hereafter called VDE-DSBs) form independent of Spo11 and meiotic axis proteins. However, like Spo11-DSBs, VDE-DSBs form after pre-meiotic DNA replication and are repaired using the same end-processing and strand invasion activities that repair Spo11-DSBs (Fukuda etal., 2003; Hodgson etal., 2011; Neale et al., 2002). We examined resolvase contributions to VDE-initiated CO formation, and obtained evidence that local enrichment for meiotic axis proteins promotes MutLγ-dependent CO formation; while recombination that occurs outside of this specialized environment forms COs by MutLγ-independent mechanisms. We also show that CO formation at a locus, and in particular MutLγ-dependent CO formation, requires Spo11-DSB formation elsewhere in the genome.
Results
Using VDE to study meiotic recombination at “hot” and “cold” loci
The recombination reporter used for this study contains a VDE recognition sequence (VRS) inserted into a copy of the ARG4 gene on one chromosome, and an uncleavable mutant recognition sequence (VRS103) on the homolog (Figure 1). Restriction site polymorphisms at flanking HindIII sites, combined with the polymorphic VRS site, allow differentiation of parental and recombinant DNA molecules. This recombination reporter was inserted at two loci: HIS4 and URA3, which are “hot” and “cold”, respectively, for Spo11-initiated recombination and Red1/Hop1 occupancy (Borde etal., 1999; Buhler etal., 2007; Pan etal., 2011; Panizza et al., 2011; Wu and Lichten, 1995); also see Figure 4A and Figure 4—figure supplement 1, below). Consistent with previous reports, Spo11-DSBs and the resulting crossovers, are five times more frequent in inserts at HIS4 than at URA3 (Figure 1—figure supplement 1A). When VDE is expressed, ~90% of VRS sites at both loci were cleaved by 7 h after initiation of sporulation (Figure 2A), consistent with previous reports that VDE cuts very efficiently (Johnson et al., 2007; Neale et al., 2002; Terentyev et al., 2010). DSBs appeared and disappeared with similar timing at the two loci (Figure 2B), with measures of insert recovery (Figure 2—figure supplement 1A) and levels of interhomolog recombinants relative to cumulative VDE-DSB levels (Figure 2—figure supplement 1B) indicating that ~70% of VDE DSBs are repaired by interhomolog recombination. The remaining VRS-containing inserts appear to be lost, consistent with high levels of VDE activity preventing recovery of inter-sister recombinants. Thus, the two VDE recombination reporter inserts undergo comparably high levels of meiotic recombination initiation, regardless of the local intrinsic level of Spo11-initiated recombination.
When VDE-DSBs are repaired by interhomolog recombination, VRS sequences are converted to VRS103, and become resistant to digestion by VDE. We therefore used HindIII/VDE double digest to score VDE-initiated recombination (Figure 1). By comparing levels of recombinants in VDE-expressing and vdeΔ strains, we determined that Spo11-initiated events make a negligible contribution to recombinants scored in VDE-expressing strains (Figure 2C, Figure 1—figure supplement 1). VDE-initiated recombinants formed at high frequencies at both HIS4 and URA3 (Figure 2C), and NCOs exceeded COs by approximately twofold at HIS4 and threefold at URA3 (Figure 2D). These values are within the range observed in genetic studies of Spo11-induced gene conversion in budding yeast (Fogel etal., 1979), but differ from the average of near-parity between NCOs and COs observed in molecular assays (Lao et al., 2013; Martini et al., 2006). VDE, unlike Spo11, frequently cuts both sister chromatids (Gimble and Thorner, 1992; Zhang et al., 2011), and this may reduce the fraction of DSBs that are repaired as COs (Malkova et al., 2000).
MutLγ makes different contributions to VDE-initiated CO formation at the two insert loci
While VDE-initiated recombination occurred at similar levels in inserts located at HIS4 and at URA3, we observed a marked difference between the two loci, in terms of the resolvase-dependence of CO formation (Figure 3). At the HIS4 locus, COs were reduced in mlh3 mutants, which lack MutLγ, by ~60% relative to wild type. COs were reduced by ~30% in in mms4-mdyen1Δ slx1Δ mutants, (hereafter abbreviated as ssn mutants), which lack the three structure selective nucleases (SSNs) active during both meiosis and the mitotic cell cycle, and by ~75% in mlh3 ssn mutants. Thus, like Spo11-initiated COs, VDE-initiated COs in inserts at HIS4 are primarily MutLγ-dependent, and less dependent on SSNs. In contrast, COs in inserts located at URA3 were reduced by only ~10% in mlh3, by ~40% in ssn mutants, and by ~60% in mlh3 ssn mutants, leaving approximately the same residual CO levels as was seen at HIS4. Thus, SSNs make a substantially greater contribution to VDE-initiated CO formation at URA3 than does MutLγ, and MutLγ’s contribution becomes substantial only in the absence of SSNs.
At both insert loci, ssn and mlh3 ssn mutants accumulated DNA species with reduced electrophoretic mobility (Figure 3—figure supplement 2). These slower-migrating species contain branched DNA molecules, as would be expected for unresolved joint molecules (D. M., unpublished observations). Steady state VDE-DSB levels and final NCO levels were similar in all strains (Figure 3D, Figure 3—figure supplement 1), indicating that resolvases do not act during the initial steps of DSB repair, and that most meiotic NCOs form by mechanisms that do not involve Holliday junction resolution (Allers and Lichten, Muyt et al., 2012; Sourirajan and Lichten, 2008; Zakharyevich et al., 2012).
Altered Hop1 occupancy in pch2 mutants is associated with altered MutLγ-dependence of VDE-initiated COs
The marked MutLγ-dependence and -independence of VDE-initiated COs in inserts at HIS4 and at URA3, respectively, are paralleled by levels of occupancy at the two loci of the meiotic axis proteins, Hop1 and Red1 (Panizza et al., 2011); Figure 4A, Figure 4—figure supplement 1A). To ask if differential Hop1 occupancy is responsible for the observed differences in CO formation at these loci, we examined the resolvase-dependence of VDE-initiated COs in pch2Δ mutants. Pch2 is a conserved AAA ATPase that maintains the nonuniform pattern of Hop1 occupancy along meiotic chromosomes (Börner et al., 2008; Joshi et al., 2009). The different Hop1 occupancies seen in wild type were preserved early in meiosis in pch2Δ mutants (Figure 4A, Figure 4—figure supplement 1A), consistent with previous findings that, in pch2 cells, Spo11-DSB patterns are not altered in most regions of the genome (Vader et al., 2011). By contrast, at later times (4-5 h after initiation of meiosis), pch2Δ mutants displayed reduced Hop1 occupancy at HIS4, more closely approaching the lower occupancy levels seen throughout meiosis at URA3 (Figure 4A; Figure 4—figure supplement 4A).
While VDE-induced DSB dynamics and NCO levels were similar in PCH2 and pch2Δ strains (Figure 4—figure supplement 1B, C), the loss of Pch2 was accompanied by a marked change in MutLγ contributions to VDE-initiated COs. The majority of COs became MutLγ-independent at both insert loci (Figure 4B, C). At HIS4, the fraction of COs that were MutLγ-dependent decreased substantially (from ~60% in PCH2 to 20% in pch2Δ), while at URA3 the fraction that were MutLγ-dependent increased (from ~10% to 37%). Thus, in pch2Δ mutants, the similarity of Hop1 occupancy at later times in meiosis is paralleled by a shift in the MutLγ-dependence of VDE-initiated COs, with contributions of MutLγ to COs in inserts at HIS4 and URA3 becoming more similar.
Spo11-DSBs promote VDE-initiated, MutLγ-dependent COs
All experiments reported above used cells with wild-type levels of Spo11-DSBs. While VDE-DSBs form at similar levels and timing in SPO11 and spo11 mutant cells (Johnson et al., 2007; Neale etal., 2002; Terentyev etal., 2010), features of VDE-DSB repair, including the extent of end resection, are strongly influenced by the presence or absence of Spo11-DSBs (Neale etal., 2002). To determine if other aspects of VDE-initiated recombination are also affected, we examined VDE-initiated recombination in a catalysis-null spo11-Y135Fmutant, hereafter called spo11. In spo11 mutants, VDE-DSB dynamics and NCO formation were similar in inserts at HIS4 and URA3, were comparable to those seen in wild type (Figure 5— figure supplement 1), and were independent of HJ resolvase activities (Figure 5—figure supplement 1). In contrast, the absence of Spo11-DSBs substantially reduced VDE-induced COs, resulting in virtually identical CO timing and levels at the two loci (Figure 5A). Unlike the ~60% reduction in COs seen at HIS4 in SPO11 mlhΔ (Figure 3C), final CO levels were similar in spo11 mlhΔ and spo11 MLH3 strains, atboth HIS4 and URA3, and similar CO reductions were observed at both loci in spo11 snn mutants (Figure 5B, C). Thus, processes that depend on Spo11-DSBs elsewhere in the genome are important to promote VDE-initiated COs, and appear to be essential for MutLγ-dependent CO formation.
Discussion
Local chromosome structure influences meiotic CO formation
We examined the contribution of different Holliday junction resolvases to VDE-initiated CO-formation in recombination reporter inserts at two loci, HIS4 and URA3, which are “hot” and “cold”, respectively, for Spo11-inititiated recombination and for occupancy by the meiotic chromosome axis proteins, Hop1 and Red1. VDE-initiated COs at HIS4 are similar to those initiated by Spo11, in that most depend on MutLγ. In contrast, VDE-initiated COs at the “cold” locus, URA3, more closely resemble mitotic COs, which are independent of MutLγ, but are substantially dependent on SSNs (Ho et al., 2010; Ira et al., 2003; Muñoz-Galván et al., 2012; Welz-Voegele et al., 2002). Locus-dependent differences in MutLγ-dependence are reduced in pch2Δ mutants, as are differences in Hop1 occupancy at later times in meiosis I prophase. Based on these findings, we suggest that local chromosome context exerts an important influence on the biochemistry of CO formation during meiosis, and that factors responsible for creating DSB-hot and -cold domains also create corresponding domains where different DSB repair pathways are dominant. An attractive hypothesis (Figure 6) is that regions enriched for meiosis-specific axial element proteins create a chromosomal environment that promotes meiotic DSB formation, limits inter-sister recombination, preferentially loads ZMM proteins (Joshi et al., 2009; Serrentino et al., 2013), and is required for recruitment of MutLγ. In such regions, where most Spo11-dependent events occur, recombination intermediates will have a greater likelihood of being captured by axis-associated ZMM proteins, and consequently being resolved as COs by MutLγ. Regions with lower axial element protein enrichment are less likely to recruit ZMM proteins and MutLγ; DSB repair and CO formation in these regions is more likely to involve non-meiotic mechanisms. In short, the meiotic genome can be thought of as being apportioned between two different environments: meiotic axis protein-enriched regions, where “meiotic” recombination pathways predominate; and meiotic axis protein-depleted regions, in which recombination events more closely resemble those seen in mitotic cells.
While the current study is the first to directly query the effect of chromosome context on JM resolution, others have obtained results that are consistent with an effect of local chromosome context on meiotic DSB repair. Malkova and coworkers used the HO endonuclease to initiate recombination in meiotic cells at LEU2, also a “hot” locus (Panizza et al., 2011; Wu and Lichten, 1995). The resulting COs were dependent on Msh4, a ZMM protein, to the same degree as are Spo11-induced COs, suggesting that these nuclease-induced COs at the axis enriched LEU2 locus (Panizza et al., 2011) were the products of ZMM/MutLγ-dependent JM resolution (Malkova etal., 2000). Consistent with our suggestion that different recombination mechanisms operate in different parts of the genome, the meiotic genome also appears to be divided into regions that respond to DNA damage in different ways. Treatment of meiotic yeast cells with phleomycin, a DSB-forming agent, triggers Rad53 phosphorylation, as it does in mitotic cells, while Spo11-DSBs do not (Cartagena-Lirola et al., 2008). This indicates that Spo11-DSBs form in an environment that is refractory to Rad53 recruitment and modification, but that there also are regions in the meiotic genome where exogenously-induced damage can trigger the mitotic DNA damage response. In light of these ideas, it is interesting that the meiotic defects of spo11 mutants in a variety of organisms are often only partially rescued by treatment with exogenous agents that cause DSBs (Bowring et al., 2006; Celerin et al., 2000; Dernburg et al., 1998; Loidl and Mochizuki, 2009; Pauklin etal., 2009; Storlazzi etal., 2003; Thorne and Byers, 1993). While other factors may be responsible for the limited rescue observed, we suggest that it reflects the random location of exogenously-induced DSBs, with only a subset forming in regions where repair is likely to form interhomolog COs that promote proper homolog segregation.
The interplay of resolvase activities is chromosome context-dependent
Although we observe marked differences in the contributions of different resolvases to VDE-induced CO formation at HIS4 and at URA3, there is no absolute demarcation between MutLγ and SSN activities at the two loci. At HIS4, where MutLγ predominates, ssn mutants still display a modest reduction in VDE-initiated COs when MutLγ is active, but an even greater relative reduction in the absence of MutLγ. These findings are consistent with previous studies suggesting that, in the absence of MutLγ, SSNs are able to serve as a backup JM resolvase (Argueso et al., 2004; De Muyt et al., 2012; Zakharyevich et al., 2012). Our current data indicate that the converse may also be true, since at URA3, MutLγ appears to make a greater contribution to CO formation in the absence of SSNs than in their presence. However, in our studies, JMs are more efficiently resolved in mlhΔ mutants than in ssn mutants, which display persistent unresolved JMs. Therefore, if MutLγ acts as a back-up resolvase, it can do so in only a limited capacity, possibly reflecting a need for a specific chromosome structural context in which to function efficiently. The absence of such a meiosis-specific chromosome context may explain why MutLγ does not appear to contribute to CO formation during the mitotic cell cycle (Ira et al., 2003; Welz-Voegele et al., 2002), although the lower expression of MLH3 expression in mitotic cells (Brar et al., 2012; Primig etal., 2000) may also contribute.
Both VDE-induced and Spo11-induced COs form at significant frequencies in mlhΔ ssn mutants, which lack all four of the HJ resolvase activities thought to function during meiosis (Figure 3, see also (Argueso etal., 2004; Zakharyevich etal., 2012). These residual crossovers may reflect the activity of a yet-unidentified JM resolvase; they may also reflect the production of half-crossovers by break-induced replication (Ho etal., 2010; Kogoma, 1996; Llorente et al., 2008) or by other mechanisms that do not involve dHJ-JM formation and resolution (Ho et al., 2010; Ivanov and Haber, 1995; Muñoz-Galván et al., 2012; Prado and Aguilera, 1995).
Genome-wide Spo11-DSBs promote VDE-initiated COs and are required for chromosome context-dependent differentiation of VDE DSB repair
In catalysis-null spo11-Y135Fmutants, most VDE-DSBs are repaired by interhomolog recombination (Figure 4, Figure 4—figure supplement 2), indicating that a single DSB can efficiently search the meiotic nucleus for homology. However, VDE-promoted COs are substantially reduced in spo11 mutants (Figure 4), as has been observed with HO endonuclease-induced meiotic recombination (Malkova et al., 2000). Moreover, in spo11 mutants, virtually all VDE-initiated COs are MutLγ-independent (Figure 4, Figure 4—figure supplement 2). Because Hop1s occupancy of cohesin sites is not noticeably altered in spo11-Y135Fmutants (Franz Klein, personal communication), these findings indicate that, in addition to the local effects of meiotic chromosome structure suggested above, CO formation is affected by processes that require Spo11-DSBs elsewhere in the genome.
Meiotic DSB repair occurs concurrently with homolog pairing and synapsis (Börner et al., 2004; Padmore etal., 1991), and efficient homolog synapsis requires wild-type DSB levels, indicating that multiple interhomolog interactions along a chromosome are needed for stable homolog pairing (Henderson and Keeney, 2004). To account for the reduced levels and MutLγ-independence of VDE-initiated COs in spo11 mutants, we suggest that a single VDE-DSB is not sufficient to promote stable homolog pairing, and that additional DSBs along a chromosome are needed to promote stable homolog pairing, which in turn is needed to form ZMM protein-containing structures that stabilize JMs and recruit MutLγ. However, the 140-190 Spo11-DSBs that form in each meiotic cell (Buhler et al., 2007; Martini et al., 2011; Pan et al., 2011) are also expected to induce a nucleus-wide DNA damage-response, and to compete with other DSBs for repair activities whose availability is limited, and both have the potential to alter recombination biochemistry at VDE-DSBs (Johnson et al., 2007; Neale et al., 2002). Thus, while we believe it likely that defects in homolog pairing and synapsis are responsible for the observed impact of spo11 mutation on VDE-initiated CO formation, it remains possible that it is due to changes in DNA damage signaling, repair protein availability, or in other processes that are affected by global Spo11-DSB levels.
Concluding remarks
We have provided evidence that structural features of the chromosome axis, in particular the enrichment for meiosis-specific axis proteins, create a local environment that directs recombination to “meiotic” biochemical pathways. In the remainder of the genome, biochemical processes more typical of mitotic recombination function. In other words, the transition to meiosis from the mitotic cell cycle does not require a global inhibition of mitotic” recombination mechanisms, which remain active in the meiotic nucleus and have the capacity to act in recombination events that occur outside of the local “meiotic “ structural context. It is well established that this local chromosome context influences the first step in meiotic recombination, Spo11-catalyzed DSB formation (Panizza et al., 2011; Prieler et al., 2005). Our work shows that it also influences the last, namely the resolution of recombination intermediates to form COs. It will be of considerable interest to determine if other critical steps in meiotic recombination, such as choice between sister and homolog as a DSB repair partner, are also influenced by local aspects of chromosome structure.
In the current work, we focused on correlations between local enrichment for the meiosis-specific axis protein Hop1 and Holliday junction resolution activity during CO formation. Other HORMA domain proteins, including HIM-3 and HTP-1/2/3 in C. elegans, ASY3 in A thaliana and HORMAD1/2 in M. musculus also have been reported to regulate recombination and homolog pairing (Kim et al., 2014; Martinez-Perez and Villeneuve, 2005) (Ferdous etal., 2012; Fukuda etal., 2010; Wojtasz etal., 2009), suggesting that HORMA domain proteins may provide a common basis for the chromosome context-dependent regulation of meiotic recombination pathways in eukaryotes.
Materials and Methods
Yeast strains
All yeast strains are of SK1 background (Kane and Roth, 1974), and were constructed by standard genetic crosses or by direct transformation. Genotypes and allele details are given in Supplementary Table 1. Recombination reporter inserts with arg4-VRS103 contain a 73nt VRS103 oligonucleotide containing the mutant VDE recognition sequence from the VMA1-103 allele (Fukuda et al., 2007; Nogami et al., 2002) inserted at the EcoRV site in ARG4 coding sequences within a pBR322-based plasmid with URA and ARG4 sequences, inserted at the URA3 and HIS4 loci, as described (Wu and Lichten, 1995). Recombination reporter inserts with the cleavable arg4-VRS (Neale et al., 2002) were derived from similar inserts but with flanking repeat sequences removed, to prevent repair by single strand annealing (Pâques and Haber, 1999). This was done by replacing sequences upstream and downstream of ARG4 with natMX (Goldstein and McCusker, 1999) and K. lactis TRP1 sequences (Stark and Milner, 1989) respectively (see Supplementary Table 1 legend for details. The resulting arg4-VRS and arg4-VRS103 inserts share 3.077 kb of homology.
VDE normally exists as an intein in the constitutively-expressed VMA1 gene (Gimble and Thorner, 1993), resulting in low levels of DSB formation in presporulation cultures (data not shown), probably due to small amounts VDE incidentally imported to the nucleus during mitotic growth (Nagai et al., 2003). To further restrict VDE DSB formation, strains were constructed in which VDE expression was copper-inducible. These strains contain the VMA1-103 allele (Nogami et al., 2002), which provides wild type VMA1 function, but lacks the VDE intein and is resistant to cleavage by VDE. To make strains in which VDE expression was copper-inducible, VDE coding sequences on an EcoRI fragment from pY2181 (Nogami et al., 2002) (a generous gift from Drs. Satoru Nogami and Dr. Yoshikazu Ohya) were inserted downstream of the CUP1 promoter in plasmid pHG40, which contains the kanMX selectable marker and a ~1kb CUP1 promoter fragment (Jin et al., 2009), to make pMJ920, which was then integrated at the CUP1 locus.
Sporulation
Yeast strains were grown in buffered liquid presporulation medium and shifted to sporulation medium as described (Goyon and Lichten, 1993), except that sporulation medium contained 10uM CuSO4 to induce VDE expression. All experiments were performed at 30°C. Independent experiments were performed either on different days, or on the same day with cultures derived from independent single colonies.
DNA extraction and analysis
Genomic DNA was prepared as described (Allers and Lichten, 2000). Recombination products were detected on Southern blots containing genomic DNA digested with HindIII and VDE (PI-SceI, New England Biolabs), using specific buffer for PI-SceI. Samples were heated to 65° C for 15 min before loading to disrupt VDE-DNA complexes; gels contained 0.5% agarose in 45 mM Tris Borate + 1 mM EDTA (1X TBE) and were run at 2 V/cm for 24-25 hours. DSBs were similarly detected on Southern blots, but were digested with HindIII alone as previously described (Goldfarb and Lichten, 2010), and electrophoresis buffer was supplemented with 4mM MgCl2. Gels were transferred to membranes and hybridized with radioactive probe as described (Allers and Lichten, 2001a; 2001b), and were imaged and quantified using a Fuji FLA-5100 phosphorimager and ImageGauge 4.22 software. HindIII-VDE gel blots were probed with ARG4 sequences from −430 to +63 nt relative to ARG4 coding sequences (Probe 1, Figure 1). HindIII gel blots were probed with sequences from the DED81 gene (+978 to +1650 nt relative to DED81 coding sequence), which is immediately upstream of ARG4 (Probe 2, Figure 2).
Chromatin immunoprecipitation and quantitative PCR
Cells were formaldehyde-fixed by adding 840 μl of a 36.5-38% formaldehyde solution (Sigma) to 30 ml of meiotic cultures, incubating for 15 minutes at room temperature, and quenched by the addition of glycine to 125 mM. Cells were harvested by centrifugation, resuspended in 500 μl lysis buffer (from (Strahl-Bolsinger etal., 1997) except with 1mg/ml Bacitracin and cOmplete Roche protease inhibitor cocktail (1 tablet/10ml) as protease inhibitors) and lysed at 4°C via 10 cycles of vortexing on a FastPrep24 (MP Medical) at 4 M/sec for 40 secs, with 5 minute pauses between runs. Lysates were then sonicated to yield an average DNA size of 300 bp and clarified by centrifugation at 21,130 RCF for 20 minutes. 1/50th of the sample was removed as input, and 2μl of anti-Hop1 (a generous gift from Nancy Hollingsworth) was added to the remainder (~490 μl) and incubated with gentle agitation overnight at 4°C. Antibody complexes were purified by addition of 20 μl of 50% slurry of Gammabind G Sepharose beads (GE healthcare), with further incubation for 3 hours at 4°C, followed by pelleting at 845 RCF for 30 seconds. Beads were then washed and processed for DNA extraction as described (Blitzblau 1993; Andreas Hochwagen, personal communication).
qPCR analysis of purified DNA from input and immunoprecipitated samples used primer pairs that amplify two regions (chromosome III coordinates 65350-65547 and 68072-68271, Saccharomyces Genome Database release # R64-2-1) flanking the HIS4 gene, and two regions (chromosome V coordinates 115119-115317 and 117728-117922) flanking the URA3 gene (see Figure 1—figure supplement 1). Primers and genomic DNA from input and immunoprecipitated samples were mixed with iQ SYBR green supermix (Biorad) and analyzed using a Biorad iCycler.
Acknowledgements
We thank Robert Shroff, Anuradha Sourirajan, Satoru Nogami, Yoshikazu Ohya, and Nancy Hollingsworth for strains and reagents, Andreas Hochwagen and Franz Klein for communicating unpublished information, and Dhruba Chattoraj, Julie Cooper, and Alex Kelly for comments on the manuscript.