Copyright Ó 2009 by the Genetics Society of America DOI: 10.1534/genetics.109.102640
Tethering Recombination Initiation Proteins in Saccharomyces cerevisiae Promotes Double Strand Break Formation Demelza R. Koehn, Stuart J. Haring,1 Jaime M. Williams and Robert E. Malone2 Department of Biology, University of Iowa, Iowa City, Iowa 52242-1324 Manuscript received March 10, 2009 Accepted for publication March 26, 2009 ABSTRACT Meiotic recombination in Saccharomyces cerevisiae is initiated by the creation of DNA double strand breaks (DSBs), an event requiring 10 recombination initiation proteins. Published data indicate that these 10 proteins form three main interaction subgroups [(Spo11-Rec102-Rec104-Ski8), (Rec114-Rec107-Mei4), and (Mre11-Rad50-Xrs2)], but certain components from each subgroup may also interact. Although several of the protein–protein interactions have been defined, the mechanism for DSB formation has been challenging to define. Using a variation of the approach pioneered by others, we have tethered 8 of the 10 initiation proteins to a recombination coldspot and discovered that in addition to Spo11, 6 others (Rec102, Rec104, Ski8, Rec114, Rec107, and Mei4) promote DSB formation at the coldspot, albeit with different frequencies. Of the 8 proteins tested, only Mre11 was unable to cause DSBs even though it binds to UASGAL at GAL2. Our results suggest there may be several ways that the recombination initiation proteins can associate to form a functional initiation complex that can create DSBs.
I
N Saccharomyces cerevisiae, meiotic recombination is initiated by the formation of DNA double strand breaks (DSBs) (e.g., Keeney 2001). These initiation events do not occur randomly along the length of each chromosome; rather, some regions (hotspots) have a higher probability of DSB formation (Sun et al. 1989; Gerton et al. 2000). Hotspots are often found in promoter regions and other areas of open chromatin and are usually absent around centromeres and telomeres (Baudat and Nicolas 1997; Petes 2001; Blat et al. 2002). A genomewide microarray analysis suggested that hotspots may be preferentially located in large (tens of kilobases) chromosomal domains of higher GC content (Gerton et al. 2000); however, there appears to be no obvious hotspot consensus sequence (Haring et al. 2004). Ten proteins [Spo11, Rec102, Rec104, Ski8/Rec103 (hereafter referred to as Ski8), Rec114, Mei4, Rec107/ Mer2 (hereafter referred to as Rec107), Mre11, Rad50, and Xrs2] are essential to produce the DSBs that initiate meiotic recombination (reviewed in Keeney 2001). The meiotic phenotypes conferred by null mutations of the initiation genes are indistinguishable: reduced sporulation, no DSBs or recombination, disrupted synaptonemal complex, and inviable spores (e.g., Malone et al. 1991). Of the 10 recombination initiation proteins, only Spo11 has been assigned a biochemical function; it
1 Present address: Department of Chemistry, Biochemistry, and Molecular Biology, North Dakota State University, Fargo, ND 58105. 2 Corresponding author: Department of Biology, University of Iowa, Iowa City, IA 52242-1324. E-mail:
[email protected]
Genetics 182: 447–458 ( June 2009)
covalently attaches to the ends of the DSBs (Bergerat et al. 1997; Keeney et al. 1997). The requirement for 10 proteins to start recombination suggests that at least some of them form a complex to make DSBs. Several analyses have demonstrated that three subcomplexes exist among the 10 recombination initiation proteins: the Spo11-subcomplex: Spo11, Ski8, Rec102, and Rec104 (Salem et al. 1999; Uetz et al. 2000; Kee and Keeney 2002; Jiao et al. 2003; Arora et al. 2004; Cheng et al. 2004; Kee et al. 2004); the Rec114subcomplex: Rec114, Rec107, and Mei4 (Arora et al. 2004; Henderson et al. 2006; Li et al. 2006; Maleki et al. 2007; Sasanuma et al. 2008); and the MRX-subcomplex: Mre11, Rad50, and Xrs2 (Borde 2007). Some data suggest that components from different subcomplexes also interact (Arora et al. 2004); one interpretation is that the 10 proteins form a single holocomplex to create DSBs. However, the exact mechanism for how these 10 proteins lead to DSB formation has been challenging to define; the literature describing their interactions contains a number of results that appear inconsistent. One example of differing observations is the relationship between the Spo11-subcomplex and the Rec114subcomplex (Prieler et al. 2005; Sasanuma et al. 2008; Wan et al. 2008). Chromatin immunoprecipitation indicated that Spo11 was not present at the ARE1 hotspot in the absence of REC114 (Prieler et al. 2005; Sasanuma et al. 2008) or phosphorylatable REC107 (Sasanuma et al. 2008). Consistent with this, Rec114 and Mei4 were present on chromatin (Maleki et al. 2007), and at hotspots (ChIP) (Sasanuma et al. 2008) in the absence of SPO11 (Sasanuma et al. 2008). However,
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D. R. Koehn et al. TABLE 1 Yeast strain list
Strains
Genotype
RM96
K264-10D MAT a lys2 -2 tyr 1-2 his7 -1 ura3 -1 HIS1 CAN 1 ADE5 met13 -c trp5 -c leu1-c cyh2 RM26-26C MAT a lys2 -1 TYR1 HIS7 ura3 -1 his1 can1r ade5 MET 13 trp5 -2 leu1-12 CYH S
DRH1-62 DRH1-54 DRH1-56 DRH1-68 DRH1-59 DRH1-109 DRH1-65 DRH1-112 DRH1-90 DRH1-81 DRH1-71 DRH1-75 DRH1-87 DRH1-78 DRH1-97 DRH1-84 DRH1-98 DRH1-121 DRH1-122 DRH1-123
Source or reference r
S
r
Isogenic to RM96; homozygous spo11DTkan Isogenic to RM96; homozygous rec102DTkanr Isogenic to RM96; homozygous rec104DTkanr Isogenic to RM96; homozygous ski8DTkanr Isogenic to RM96; homozygous rec114DTkanr Isogenic to RM96; homozygous rec107DTkanr Isogenic to RM96; homozygous mei4DTkanr Isogenic to RM96; homozygous mre11DTkanr Isogenic to RM96; homozygous rad50STURA3 Same as DRH1-62 except rad50STURA3/rad50STURA3 Same as DRH1-54 except rad50STURA3/rad50STURA3 Same as DRH1-56 except rad50STURA3/rad50STURA3 Same as DRH1-68 except rad50STURA3/rad50STURA3 Same as DRH1-59 except rad50STURA3/rad50STURA3 Same as DRH1-90 except rec107DTkanr/rec107DTkanr Same as DRH1-65 except rad50STURA3/rad50STURA3 Same as DRH1-90 except mre11DTkanr/mre11DTkanr Same as DRH1-81 except gal4DTTRP5/gal4DTTRP5 Same as DRH1-97 except gal4DTTRP5/gal4DTTRP5 Same as DRH1-98 except gal4DTTRP5/gal4DTTRP5
Prieler et al. (2005) found that Spo11 binds hotspot DNA in both a rec107D and a mei4D strain. These articles come to different conclusions about the interactions of Spo11 and the Rec114-subcomplex. Naturally occurring recombination hotspots [e.g., ARE1 (formerly YCR048W), ARG4, CYS3, and HIS2] have been useful to study the interactions of recombination initiation proteins with S. cerevisiae chromosomes (e.g., Lichten and Goldman 1995; Prieler et al. 2005; Sasanuma et al. 2007) and with each other. A complementary approach to the analysis of natural hotspots, attracting recombination proteins to a site that was previously cold, was devised by Pecin ˜a et al. (2002). They demonstrated that the Gal4 DNA binding (DB) domain fused to Spo11 efficiently promoted recombination initiation at UASGAL sites normally cold for recombination. For example, DSB formation at GAL2 increased from undetectable levels to 12% of total DNA (Pecin ˜a et al. 2002). The new Gal4DB-Spo11-dependent hotspot was also completely dependent on the presence of all nine of the other initiation proteins. Although Spo11 is the putative DSB catalyst, it alone was incapable of producing DSBs, even when brought to the DNA by the Gal4 DB domain. These results suggest that Spo11 was able to recruit all the other required proteins to the coldspot. Given the complex network of interactions among the recombination initiation proteins, we thought it was probable that some initiation proteins would be capable of inducing DSBs when tethered to a coldspot, while others would not. By comparing which
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proteins could make DSBs vs. those which could not, our goal was to gain a better understanding of how a functional initiation complex was assembled on the DNA. We tethered 8 of the 10 recombination initiation proteins to UASGAL sites by using a slight modification of the Pecin ˜a et al. (2002) approach. We demonstrate that, in addition to Spo11, six other Gal4DB fusions (DBRec102, DB-Rec104, DB-Ski8, DB-Rec114, DB-Rec107, and DB-Mei4) are able to cause DSBs upstream of the naturally cold GAL2 gene. Proteins capable of stimulating initiation include components from the Spo11subcomplex and the Rec114-subcomplex. We are unable to detect DSB formation when a protein from the MRX-subcomplex (Mre11) is tethered.
MATERIALS AND METHODS Yeast strains: The yeast strains used in these experiments were derived from the diploid RM96 (S288C background), whose genotype is listed in Table 1. The relevant genetic markers of all other strains derived from it are listed (Table 1). Recombination mutations were constructed by gene replacement using standard one-step gene replacement with PCR fragments amplified from the Research Genetics strain collection (Giaever et al. 2002). Diploids containing the RAD50 wild-type allele were used to test fusion constructs by complementation tests; diploids containing the rad50S mutation were used for the analysis of DSBs (see below). Plasmid construction: All GAL4DB-HA-REC fusion constructs, where ‘‘REC’’ represents any of eight recombination initiation genes, were constructed with the YEplac195 2m-vector (Gietz and Sugino 1988). These plasmids were constructed
Meiotic Recombination Initiation
Figure 1.—Construction of fusion plasmids. All GAL4DBREC fusion plasmids were constructed similarly by cloning the coding region of each recombination initiation gene into NheI and SalI sites of a YEplac195-based vector, placing each REC gene in frame with an HA-tagged copy of the GAL4 DNA binding (DB) domain. These constructs were driven by the ADH1 promoter and terminated by the ADH1 transcriptional terminator (see materials and methods). All constructs were confirmed by DNA sequencing.
as follows: A REC104-containing fragment was removed from pAMG406 (Salem et al. 1999) using PvuII and religated to make pSJH17. A ScaI–PvuII fragment containing pADH1GAL4DB-HA-REC104-tADH1 from pYM9D-1 was then ligated into the PvuII site of pSJH17 to make pSJH30 (Figure 1). pDRH2 was made by removing the NheI–SalI fragment containing REC104 from pSJH30. pDRH1, pDRH3, pDRH4, pDRH5, pDRH7, pDRH8, and pSJH23 were made by inserting a PCR fragment containing the coding region of the appropriate REC gene into the NheI and SalI sites pSJH30. The PCR primers were engineered such that the final PCR product contained an upstream NheI site and a downstream SalI site. pSJH22 was constructed similarly, except the final PCR product contained NheI and PstI restriction enzyme sites. pDRH6 was made by subcloning a KpnI–XbaI MEI4-containing fragment from pBluescript SK1 (Menees et al. 1992) into pRS316 (Sikorski and Hieter 1989). All fusion constructs were sequenced to verify that the construction was correct. All plasmids used in this study are listed in Table 2. Complementation of fusion plasmids: For each GAL4DBHA-REC fusion, the appropriate Rec diploid was separately transformed with one of three plasmids: the GAL4DB-HA vector (pDRH2), the wild-type (WT) REC plasmid, or the appropriate GAL4DB-HA-REC construct. Transformed diploids were grown on SC Ura plates, replica plated to 2% acetate sporulation plates, and incubated at 30° for 4–5 days. Sporulation plates were then replicated to media diagnostic for gene conversion (SC Leu, SC Lys, and SC Trp) and crossing over (SC 1Can/Cyh). Both can1r and cyh2 r are recessive drug resistant markers. In a normal meiosis, 25% of spores are resistant to both drugs because of random segregation in the reductional division. A proper reductional division is dependent on proper amounts of crossing over. Rec initiation mutants have no crossing over so the frequency of doubly drug-resistant products is equal to the background
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mitotic recombination frequency (108). Complementation of the mutant Rec defect restores meiotic recombination and proper meiotic segregation, resulting in increases of haploid products expressing both drug resistances. Cells from the sporulation plates were dissected to determine viability of the meiotic products and sporulation was counted ($200 cells were counted for each of 2–4 independent diploids). Analysis of DSBs: To determine the frequency of DSB formation at ARE1 and GAL2, the rad50KI81 (Cao et al. 1990) (hereafter referred to as rad50S) mutation was used. rad50STura3D strains were made by one-step gene replacement of haploid strains using a rad50STURA3 PCR fragment. Removal of the URA3 selectable marker was done by cotransforming a PCR fragment of wild-type DNA downstream of RAD50 with YEp426 (Ma et al. 1987). Transformants were selected by using SC Lys media and rad50S strains lacking the URA3 selectable marker were identified by growth on 5-FOA. All rad50S transformants were verified by MMS testing, PCR testing, and Southern analysis as previously described (Mao-Draayer et al. 1996). Yeast meiotic DNA for analysis of DSBs was isolated as in Bullard et al. (1996) except with the following change: DNA back extractions were performed on all samples after the two phenol chloroform isoamyl (PCI) purifications. For the Southern analyses at GAL2, a 1-kb upstream probe was used (see results). As a control, Southerns were repeated for four DB-REC fusions using a smaller probe (0.4 kb) and the same results were obtained (data not shown). Imaging was performed using a Molecular Dynamics and/or Quantity One phosphorimager and all quantifications were performed on the 15-hr meiotic samples using Quantity One software. The 15-hr time point was used because DSBs plateau at that time; the amount of DSBs falls slightly at later times, presumably due to degradation. For GAL2 blots, the close proximity of A with B and C with D prevented quantification of individual bands, so we therefore quantified bands A and B together, and C and D together. All DSB frequencies are expressed as percentages relative to the total band intensity and were corrected for background. DSB percentages were also corrected for plasmid loss. For statistical analyses, the Student t-test was used. Chromatin immunoprecipitation: Chromatin immunoprecipitation was performed utilizing a protocol modified slightly from (Goldfarb and Alani 2004; Prieler et al. 2005); all solutions were as described in these articles except for the protease inhibitor cocktail (1 mm e-aminocaproic acid, 1.5 mm aprotinin, 2 mm benzamidine, 1 mm leupeptin, 2 mm pepstatin, and 1 mm PMSF). Samples were treated with a Fisher Scientific pulsing vortex mixer at 3000 rpm for 15 min at 4°. Lysates were sonicated with a model 150 Artek sonic dismembrator sonicator (50% output; 4 3 10-sec pulses) to fragment the chromatin. To 50 ml of whole cell extract (WCE), 200 ml of 13 TE, 1% SDS was added and samples stored at 4° until reversal of the crosslinks. For immunoprecipitation (IP), a-Mre11 antibody (Rockland no. 401-872) was added to WCE to a final concentration of 2.5 mg/ml and incubated overnight at 4°. IPs were incubated with Protein A magnetic beads (Dynal, Invitrogen) for 2 hr at 4°. Washes were as described in Prieler et al. (2005) and elutions were as in Goldfarb and Alani (2004). After crosslinks were reversed, DNA was purified with PCI extraction and ethanol precipitation. The final DNA pellets (WCE and IP DNA) were resuspended in 50 ml 13 TE. Real-time PCR was performed using PP1 [216-bp product upstream of the GAL2 UASGAL sites (targeted hotspot)] and PP2 [159-bp product, 16 kb downstream of GAL2 at CSF1 (a coldspot)] and carried out on a Roche LightCycler480 using SYBR Green I Master mix (Roche). PCR primers were used at a final concentration of 20 pmol per primer. Primer sequences are: PP1F-ACGCGACAGTAAAAGCAGCA; PP1R-CAAGTTGA GTGCGGCTACCT; PP2F-ACGTGGTGTTCCATTGACACAC;
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D. R. Koehn et al. TABLE 2 Plasmid list
Plasmids YEp426 pRS316 pAMG406 pSJH17 pYM9D-1 pSJH30 pDRH2 pJK67 pDRH5 pCM212 pDRH1 pJMG4.3H3/Sp pDRH3 pDP22 pSJH22 pCM706 pDRH8 pDRH6 pDRH4 pLRVII-15 pDRH7-10
Genotype
Source or reference
pBR322-based; Ampr, Tetr, 2m, LYS2 Shuttle vector; Ampr, CEN6, ARS4, URA3 YEplac195 1 2.8-kb HindIII–BamHI fragment (REC104) from pKMC16 pAMG406 with PvuII fragment containing REC104 removed SmaI–SalI fragment from pMA424 containing REC104 sub-cloned into pAS1 (Gal4DB expression vector) ScaI–PvuII fragment containing pADH1-GAL4DB-HA-REC104-tADH1 subcloned from pYM9D-1 into pSJH17 pSJH30 with an NheI–SalI fragment containing REC104 removed to make pADH1-GAL4DB-HA-tADH1 pJK72 cut with SmaI and SwaI and religated, leaving SPO11 as the only ORF inserted in YEp24 NheI–SalI SPO11 fragment cloned into pSJH30 to make pADH1-GAL4DB-HA-SPO11-tADH1 REC102 in pRS316 NheI–SalI REC102 fragment cloned into pSJH30 to make pADH1-GAL4DB-HA-REC102-tADH1 HindIII–SpeI fragment containing SKI8 subcloned from p6.5H3L into pRS316 NheI–SalI SKI8 fragment cloned into pSJH30 to make pADH1-GAL4DB-HA-SKI8-tADH1 REC114 lacking 116 bp intron cloned into pRS316 NheI–PstI REC114 fragment cloned into pSJH30 to make pADH1-GAL4DB-HA-REC114-tADH1 4.4-kb EcoRI–ClaI fragment containing REC107 cloned into pRS316 NheI–SalI REC107 fragment cloned into pSJH30 to make pADH1-GAL4DB-HA-REC107-tADH1 KpnI–XbaI fragment containing MEI4 subcloned from pBluescript SK1 into pRS316 NheI–SalI MEI4 fragment cloned into pSJH30 to make pADH1-GAL4DB-HA-MEI4-tADH1 SacI–HindIII fragment containing MRE11 subcloned into pRS317 NheI–SalI MRE11 fragment cloned into pSJH30 to make pADH1-GAL4DB-HA-MRE11-tADH1
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PP2R-CGCTCGTTTCCATAGTAGCCAT. For immunoprecipitated DNAs, 1/50 of the final volume was used in the PCR reactions. For WCE DNAs, 1 ml from 1:100, 1:500, and 1:2500 dilutions was used to create standard curves. All final melting curves showed a single DNA product. The amount of precipitated (IP) DNA was normalized to WCE and the results are expressed as a ratio (Borde et al. 2004) of GAL2 UASGAL hotspot vs. CSF1 coldspot.
RESULTS
GAL4DB fusion constructs complement recD diploids: To examine the ability of different initiation proteins to attract the others to a coldspot, we made fusion proteins with Gal4DB. All fusions placed the GAL4 DNA binding (DB) domain at the amino-terminal end of the eight recombination initiation genes (SPO11, REC102, REC104, SKI8, REC114, MEI4, REC107, and MRE11) (Figure 1). We tested the ability of GAL4DB-recombina-
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tion initiation protein fusion constructs (generally referred to below as DB-REC) to complement the corresponding recombination gene deletion (recD) using five metrics: spore viability, sporulation percentage, heteroallelic recombination (gene conversion), crossing over, and DSB formation at a natural hotspot. Each construct was transformed into diploids carrying null mutations (deletions of the open reading frames) of the WT gene (e.g., the DB-REC104 construct was transformed into rec104D/ rec104D cells). Each Rec diploid was also independently transformed with a plasmid containing only GAL4DB (pDRH2 vector) as a negative control or a REC1 WTcopy (i.e., no fusion) of the corresponding recombination gene as a positive control. The RM96 WT diploid (Rec1 for all initiation genes) sporulated at 88% and produced spores that were 96% viable (Table 3). Rec diploids with the corresponding WT gene on a plasmid sporulated almost as well as the
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TABLE 3 Sporulation and viability of DB-REC diploids compared to WT and recD diploids Gene D WT spo11
rec102
rec104
ski8
rec114
rec107
mei4
mre11
Plasmid None Vector SPO11 DB-SPO11 Vector REC102 DB-REC102 Vector REC104 DB-REC104 Vector SKI8 DB-SKI8 Vector REC114 DB-REC114 Vector REC107 DB-REC107 Vector MEI4 DB-MEI4 Vector MRE11 DB-MRE11
Spor (%)a
Via (%)
No. sporesb
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
96 0 87 99 0 79 85 0 92 87 0 83 94 0 83 81 2 89 80 4 91 87 0 90 88
71 64 68 70 64 82 65 60 96 122 40 144 144 64 64 105 62 72 65 52 64 144 72 90 78
88 26.8 77.7 74.6 13.6 68.3 65.3 35.8 67.8 68.1 9.5 71.0 61.2 38.2 62.8 57.9 15.5 67.5 59.5 14.8 68.8 74.0 24.0 68.1 59.5
2.8 2.1 2.3 5.0 0.2 6.3 2.3 2.8 3.6 8.0 0.8 3.5 2.7 4.7 5.6 3.4 0.7 0.7 0.7 3.1 3.1 4.1 1.4 0.4 2.1
WT refers to the Rec1 RM96 diploid. Vector refers to the Rec (Rad501) diploid containing the GAL4DB vector plasmid. The wild-type gene name (e.g., SPO11 or REC114) refers to a Rec (Rad501) diploid with the WT gene on a plasmid. DB-REC refers the Rec (Rad501) diploid containing the fusion plasmid. For strains and plasmids, see Tables 1 and 2 and Figure 2. a Sporulation percentage reflects the average 6 standard deviation from 2 to 3 independent diploids. At least 200 cells were counted for each diploid. b Number of spores dissected for analysis of viability. All tetrads dissected contained 4 spores; in ,3% of the tetrads, 2 or 3 spores stuck together and could not be separated. In these cases only the single spores were counted.
RM96 WT diploid and produced spores with 85% viability (Table 3). In contrast, cells with only the vector present had much lower sporulation and produced almost no viable spores. Sporulation and spore viability increased in every DB-Rec fusion construct compared to the vector control and the values were similar to those of cells containing the WT recombination genes. This implies that all DB-Rec fusions restore overall meiotic recombination to normal levels. To verify that the DB-REC fusion constructs restored meiotic recombination, a replica plating assay diagnostic for gene conversion and, indirectly, for crossing over, was used (Figure 2). Only background (i.e., mitotic) levels of recombination were observed in all Rec diploids (e.g., Figure 2, top row). All WT REC plasmids conferred levels of recombination similar to the wild-
Figure 2.—Complementation tests of GAL4DB-REC fusion genes for meiotic recombination. All fusion plasmids were transformed into the appropriate Rec diploid. Gene conversion was measured at heteroallelic loci (LEU1, LYS2, and TRP5) and crossing over at CAN1 and CYH2 (see materials and methods). ‘‘rec (vector)’’ is a rec107D diploid containing pDRH2 (GAL4DB). Only one recD diploid is shown since all exhibit no meiotic recombination. ‘‘WT (vector)’’ is the Rec1 diploid, RM96, containing pDRH2. The REC genes listed are the wild-type REC plasmids transformed into the corresponding null diploids (pJK67 in DRH1-62, pCM212 in DRH1-54, pAMG406 in DRH1-56, pJMG4.3H3/Sp in DRH1-68, pDP22 in DRH1-59, pCM706 in DRH1-109, pDRH6 in DRH1-65, pLRVII-15 in DRH1-112). The DB-REC plasmids listed are the fusion constructs transformed into the corresponding null diploids (pDRH5 in DRH1-62, pDRH1 in DRH1-54, pSJH30 in DRH1-56, pDRH3 in DRH1-68, pSJH22 in DRH1-59, pDRH8 in DRH1-109, pDRH4 in DRH1-65, pDRH7 in DRH1-112). N/A, not applicable—gene conversion at LYS2 could not be measured with WT MRE11 because the selectable marker for that plasmid is the LYS2 gene.
type RM96 diploid. As predicted by the sporulation and viability values, all fusion constructs promoted wild-type levels of meiotic gene conversion (at LEU1, LYS2, and TRP5) and crossing over (monitored by can1r and cyh2 r) (Figure 2). Although it was clear from the above that overall meiotic recombination occurred at high levels, we also examined DSB formation at the ARE1 hotspot (Figure 3A). The DB-REC fusion constructs were each transformed into recD rad50S diploids otherwise isogenic to the diploids used for the other complementation tests.
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Figure 3.—Gal4DB-Rec fusion proteins promote DSB formation at the natural ARE1 hotspot. (A) Schematic showing indirect end labeling of the 5.9-kb region at ARE1. Numbers represent the distance (in base pairs) relative to the 11 start codon of the gene. The arrow indicates the DSB hotspot site in that region. Asterisks (*) within ARE1 indicate UASGAL consensus sequences. (B) All samples were taken from cells at 15 hr in sporulation. (Left) Both the Rec and WT control diploids (DRH1-90) contain the pDRH2 vector. (Right) DSB analysis of GAL4DB-REC fusion plasmids in Rec diploids (pDRH5 in DRH1-81, pDRH1 in DRH1-71, pSJH30 in DRH175, pDRH3 in DRH1-87, pSJH22 in DRH1-78, pDRH8 in DRH1-97, pDRH4 in DRH1-84, pDRH7 in DRH1-98). Labels: 5.9 kb, the parental band from a HindIII restriction digest; horizontal arrow (4), hotspot DSB band; asterisks (*), DSB bands at internal UASGAL sites (see discussion); N, band not specific to meiosis. Values listed underneath each lane is the average of two experiments (6 standard deviation) at 15 hr in meiosis and corrected for plasmid loss as described in materials and methods.
Only background levels of DSBs were detected in the absence of any recombination initiation protein (e.g., see rec107D and mei4D, Figure 3B). In a Rec1 control (DRH1-90 containing pDRH2 vector), 12.3% DSBs were observed at ARE1 (Figure 3B). DSBs were detected with all fusion proteins; however, the DSB frequency varied among them (Figure 3B). In recD diploids, high DSB percentages were observed at ARE1 with DB-Ski8, DBSpo11, DB-Rec102, and DB-Rec104, consistent with previously published ARE1 values (Baudat and Nicolas 1997; Pecin ˜a et al. 2002). Lower levels were observed for DB-Mei4, DB-Rec107, DB-Rec114, and DB-Mre11, but lower percentages of DSBs at ARE1 do not necessarily imply that the fusion protein is less functional overall.
Figure 4.—DSBs at GAL2 with a Gal4DB-Spo11 fusion protein. (A) (Top) Schematic showing indirect end labeling of the region around GAL2. Numbers represent distances (in base pairs) relative to the 11 start codon of GAL2. Vertical arrows (labeled D, C, B, A) indicate DSBs. Upstream probe is the probe used to analyze KpnI-digested DNA; 39 probe is the probe used to analyze DNA digested with NcoI and XbaI (as in Pecin ˜a et al. 2002). (Bottom) Diagram (to scale) showing the positions of the DSB sites (A, B, C, and D) and the UASGAL sites (1, 2, 3, and 4). Numbers above the DSB sites indicate the base pairs upstream of the 11 ATG of GAL2. Positions of UASGAL sites: 1, 530 to 514; 2, 416 to 400; 3, 397 to 382; 4, 333 to 312. The UASGAL at ‘‘4’’ is composed of two overlapping consensus sequences, offset by 5 bp. (B) Southern blot of KpnI-digested DNA hybridized with the upstream probe. Labels above lanes: spo11 represents a spo11D diploid (DRH1-81) transformed with the GAL4DB vector (pDRH2); SPO11 is DRH1-81 transformed with pJK67; DB-SPO11 is DRH1-81 transformed with pDRH5 (two independent diploids are shown). The numbers 0, 6, and 15 refer to hours in sporulation of the sample. Labels: 2.0 kb, parental band; A, B, C, D, DSB bands; M, size markers (2.04, 1.58, 1.41, 1.14, 1.02 kb). (C) DNA from DB-SPO11 cells digested with NcoI and XbaI and probed with the 39 probe as in Pecin ˜a et al. (2002). Lanes shown are from 0 and 15 hr in sporulation. 3.7 kb, parental band; N, nonspecific bands; D, C, B, A, DSB bands.
Robine et al. (2007) demonstrated that the addition of the Gal4DB moiety to Spo11 altered the relative affinity of DB-Spo11 for different natural hotspots; overall meiotic recombination was high, but the relative frequencies of hotspots changed. This observation is likely to be true for the other DB-Rec fusion proteins we examined, since we know that overall recombination was high, as was spore viability. Taking all the data together, we conclude that the fusion proteins are capable of interacting properly with the other recombination initiation proteins to initiate meiotic recombination. Several recombination initiation proteins are able to promote DSBs at GAL2: As a control, we examined DSBs produced at GAL2 in a spo11D rad50S diploid containing the GAL4DB vector, the WT SPO11 plasmid, or
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Figure 5.—Southern blot analysis of DSBs at GAL2 created by the fusions. Labels are as in Figure 4B. (A) Indirect end labeling of GAL2 in WT cells (DRH1-90 with pDRH2 vector) and in rec104 cells. r104 is pDRH2, REC104 is pAMG406, and DBR104 is pSJH30, all present in DRH1-75. (B) Analysis of DSBs from other DB-Rec fusions. The name above the lanes represents the fusion construct present in a homozygous recD rad50S diploid. DB-SPO11 is pDRH5 in DRH181, DB-REC102 is pDRH1 in DRH1-71, DB-REC104 is pSJH30 in DRH1-75, DB-SKI8 is pDRH3 in DRH1-87, DB-REC114 is pSJH22 in DRH1-78, DB-REC107 is pDRH8 in DRH1-97, DB-MEI4 is pDRH4 in DRH1-84, and DBMRE11 is pDRH7 in DRH1-98. (C) The DNA samples (15 hr) of each diploid strain were analyzed on the same gel to compare the break locations.
the DB-SPO11 fusion plasmid. Levels of DSBs significantly above background were not observed with either of the control plasmids (Figure 4B), but the DB-fusion protein caused DSBs upstream of GAL2 at a total frequency of 15% (6 3%) (Figure 4B and later figures). This result is similar to the 12% 6 2% detected by Pecin ˜a et al. (2002). Figure 4 demonstrates that there are four DSB bands (Figure 4B). This is suggestive, since there are several UASGAL consensus sequences upstream of GAL2 (Figure 4A) (Vashee et al. 1993) (see discussion). Our experiments utilized a probe upstream of GAL2 (Figure 4A), whereas Pecin ˜ a et al. (2002) used a downstream probe and observed only one DSB (Pecin ˜ a et al. 2002). We repeated the experiment using a probe at the 39 end of the GAL2 gene (Figure 4A). The results in Figure 4C demonstrate the same four bands, except that the banding pattern, as expected, is in the opposite orientation. To determine if other initiation proteins were able to promote DSB formation at the GAL2 coldspot, we measured DSBs at GAL2 for seven DB-Rec fusion proteins (DB-Rec102, DB-Rec104, DB-Ski8, DB-Rec114, DB-Rec107, DB-Mei4, and DB-Mre11). In every experiment, neither the WT REC control plasmid nor the negative control (vector) displayed DSBs above background at GAL2 (e.g., Figure 5A and data not shown). This confirms that the GAL2 region is naturally a coldspot for recombination, and that DSB formation is dependent on tethering a recombination initiation protein to GAL2. Six initiation proteins (DB-Rec102, DB-Rec104, DB-Ski8, DB-Rec114, DB-Rec107, or DBMei4), in addition to DB-Spo11, could induce DSBs
upstream of the GAL2 coldspot (Figure 5, B and C). Only DB-Mre11 produced insignificant levels (compared to background, P ¼ 0.18) of DSB formation at GAL2 (Figure 5B; Table 4). These data indicate that seven of the eight fusion proteins tested can recruit all other required initiation proteins to the region where they are tethered. Although seven fusions can create DSBs, they do so with different frequencies (Table 4). DSBs at GAL2 in gal4D diploids: GAL4 is a positive transcriptional activator of the galactose-metabolizing genes. It is constitutively expressed in cells with and without galactose, and binds constitutively to UASGAL sites upstream of the regulated genes (Giniger et al. 1985; Huibregtse et al. 1993). It was therefore possible that the different frequencies of DSBs observed in cells with the various fusion constructs reflected, at least partially, varying abilities of the fusion proteins to compete with native Gal4 for binding to the UASGAL sequences at GAL2. This possibility was addressed by removing cellular Gal4 protein by a gal4D mutation and measuring DSBs at GAL2 in gal4D diploids containing DB-Spo11 (with high levels of DSBs) or DB-Rec107 (with low levels of DSBs) fusions. In these gal4D experiments, the same DSB bands (A, B, C, D) were observed as in previous experiments, but the amount of DSBs increased similarly for both DB-Spo11 and DB-Rec107 (1.6- and 1.4-fold, respectively; Figure 6; Table 5). These data indicate that there is some competition between the DB-Rec and Gal4 proteins, but suggests that the competition is similar for all fusion proteins. Thus, failure to compete with Gal4 protein is not the reason
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D. R. Koehn et al. TABLE 4
Quantification of DSB formation at GAL2 with DB-REC fusions in recD diploids
Relevant genotype DB-SPO11 DB-REC102 DB-REC104 DB-SKI8 DB-REC114 DB-REC107 DB-MEI4 DB-MRE11
recD diploid at GAL2: Avg. DSB (%) at 15 hr No. exp. 4 3 3 3 3 4 4 4
A 1 Ba,c 14.2 5.5 10.3 3.4 2.8 1.2 4.7 ,0.4
C 1 Da,c
Totalb,c
1.1 1.8 2.1 1.1 0.2 1.5 1.6 NDd
15.4 6 3.3 7.3 6 1.6 12.4 6 1.5 4.4 6 1.5 3.0 6 0.6 2.8 6 0.7 6.3 6 0.9 ,0.4
Strains and plasmids are as in Figure 5. a A 1 B and C 1 D are the sums of DSBs observed with bands A and B, C and D, respectively. b Total is the sum of DSBs at A, B, C, D and is the average of the number of experiments indicated 6 standard deviation. c All DSB values have been corrected for plasmid loss as described in materials and methods. d No detectable DSBs.
that the DB-Rec107 fusion creates fewer DSBs than the DB-Spo11 fusion at GAL2. DSB formation at GAL2 in Rec1 cells containing DBRec fusion proteins: For recombination initiation components suggested to normally function as multimers (Arora et al. 2004; Sasanuma et al. 2007; Fukuda et al. 2008), we reasoned that addition of the wild-type initiation protein might help to better assemble the complex. To provide the WT protein, we performed the experiments in a Rec1 strain (Figure 7) so that both DBRec protein and wild-type Rec protein were present in the cell. Four DSB bands (Figure 7) were detected at the same positions as observed in the previous experiments. As in Figure 5, there were differences in the frequency of DSBs detected with the various fusion proteins (Table 6). The ratio of DSBs observed upstream of GAL2 in the Rec vs. the Rec1 background is close to 1.0 for DB-Ski8, DB-Rec107, and DB-Mre11, indicating that the presence of the WT protein did not affect the ability of initiation to occur (Table 6). In contrast, the WT protein significantly increased DSB formation for DB-Rec102 and DB-Rec114 (P ¼ 0.02 and P , 0.01, respectively). We observed significant decreases in DSBs for DB-Mei4, DB-Spo11, and DB-Rec104 (P ¼ 0.03, P ¼ 0.04, and P , 0.01, respectively). The presence of WT protein did not help the DB-Mre11 construct. One interpretation for how the WT protein might decrease DSBs at GAL2 is if the WT protein preferentially sequesters the DB-Rec fusion or other initiation proteins at natural hotspots. DB-Mre11 binds GAL2 UASGAL sites: The only fusion protein which could not make DSBs at GAL2 was DBMre11. A possible explanation for this is that the fusion protein does not effectively bind to the UASGAL at GAL2,
Figure 6.—DSB analysis in gal4D diploids. DNA was collected at the times shown above the lanes. Labels across the top: DB-SPO11 is pDRH5 in DRH1-121, DB-REC107 is pDRH8 in DRH1-122, and DB-MRE11 is pDRH7 in DRH1-123. Markers are as in Figure 4B.
either because it cannot compete with native Gal4 for binding or because the Gal4DB moiety can not function. Since the data from Table 6 indicate that the Gal4 protein competes with DB-Spo11 and DB-Rec107, we examined the effect of removing Gal4 on the ability of DB-Mre11 to make DSBs at GAL2. We found that DSBs still do not form when native Gal4 is eliminated (Figure 6). To determine if DB-Mre11 is capable of binding at GAL2, we performed chromatin immunoprecipitation (ChIP) on mre11D diploids containing either the GAL4DB vector or the DB-MRE11 fusion construct. Two regions were monitored by PCR (Figure 8): one near the UASGAL region of GAL2, and one 16 kb downstream of GAL2. The latter region has no consensus UASGAL sites, is void of DSBs (Gerton et al. 2000), and has a physical map: genetic map ratio (Saccharomyces Genome Database) indicative of low levels of recombination. Our results indicate that DB-Mre11 does bind GAL2 UASGAL DNA during meiosis (Figure 8). At the onset of meiosis, enrichment at the targeted hotspot is the highest, is followed by an initial drop at 4 hr into sporulation, and then a steady increase as meiosis proceeds. Since DBMre11 is expressed in both mitosis and meiosis, it seems likely that much of the DB-Mre11 present in the cells at 0-hr time binds to UASGAL sites, but that after meiosis has commenced, some of the fusion protein becomes recruited to other hotspots for meiotic recombination. The data confirm that DB-Mre11 does associate with GAL2 promoter DNA, indicating the lack of DSBs observed with this fusion is not due to an inability to bind. DISCUSSION
Seven DB-Rec fusion proteins create DSBs at GAL2 at different frequencies: Ten proteins initiate meiotic recombination, but how these 10 proteins interact with each other, or with the DNA, to initiate DSB formation is still not known. It is not clear if all 10 proteins simultaneously form the initiation complex on the DNA, if a sequential assembly occurs on the DNA, or if assembly
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TABLE 5 Quantification of DSB formation at GAL2 with DB-REC fusions in recD gal4D diploids GAL2 Plasmid genotype DB-SPO11 DB-REC107
a
recD
recD gal4Da
gal4D:GAL4b
15.4 6 3.3 2.8 6 0.7
25.4 6 3.2 3.9 6 0.3
1.6 1.4
Strains and plasmids are as in Figure 6. a Numbers listed are the average (6 standard deviation) DSB percentages at 15 hr in meiosis obtained from two experiments. For recD data, see Table 4. b Numbers listed are the ratio of DSBs obtained in recD gal4D vs. recD GAL4 diploids.
occurs off the DNA. The results here indicate that tethering 7 different initiation proteins to a coldspot causes initiation to occur; even the least effective fusion created DSBs at frequencies greater than most natural recombination hotspots (Sun et al. 1991; De Massy et al. 1994; Haring et al. 2003). Of the 8 initiation proteins tested, only the DB-Mre11 fusion was unable to induce significant DSBs at the coldspot. Since Borde et al. (2004) has shown that Mre11 associates with hotspot chromatin in meiosis prior to the formation of DSBs, it was surprising to us that DB-Mre11 cannot target DSBs at GAL2. The complementation tests indicate that the DB-Mre11 fusion protein is fully functional, restoring wild-type levels of meiotic recombination and viability in an mre11D mutant. One possibility is that the binding domain of DB-Mre11 is occluded and cannot bind to UAS. The ChIP data indicate that this is not true. An alternative possibility is that once tethered, the Mre11 protein is in an orientation that prohibits an appropriate association with other required components. A third explanation is that the MRX-subcomplex normally only interacts with the other initiation proteins after components of the other two subcomplexes are associated with the DNA. This possibility is supported by data indicating that the association of Mre11 with chromatin is dependent upon the presence of all initiation proteins except Rad50 protein (Borde et al. 2004). If this explanation is correct, it suggests that DBXrs2 and DB-Rad50 would also be unable to recruit the necessary components to make breaks in this assay system. Our data indicate the eight fusion proteins examined cause different amounts of DSB formation. The frequency of DSB formation (from highest to lowest) at GAL2 was: Spo11, Rec104, Rec102, Mei4, Rec103, Rec114, Rec107, Mre11. One explanation for the DSB differences is that the DB domain fused with the Rec proteins might be differentially occluded from GAL2 UAS sites. We find no clear correlation, however, between the size of the recombination protein in the fusion and the amount of DSBs [e.g., Spo11 is 45 kDa (15.4% DSBs); Rec107 is 36 kDa (2.8% DSBs)]. Alter-
Figure 7.—Southern hybridization showing DSBs at GAL2 in Rec1 cells containing fusion constructs. The name above the lanes represents the fusion construct present in a homozygous rad50S diploid, Rec1 at all loci. DB-SPO11 is pDRH5, DB-REC102 is pDRH1, DB-REC104 is pSJH30, DB-SKI8 is pDRH3, DB-REC114 is pSJH22, DB-REC107 is pDRH8, DBMEI4 is pDRH4, and DB-MRE11 is pDRH7, all in DRH1-90. Other labels are as in Figure 4B.
natively, if an initiation complex forms off the DNA prior to binding, then the DB moiety in each fusion would be located in a different environment. For example, fusion proteins normally situated toward the center of the complex could have the DB occluded by other proteins, thereby reducing DSBs at GAL2. A second explanation for different DSB frequencies is that some DB-Rec fusions cannot compete with native Gal4 as effectively. Differential Gal4 competition could occur whether the DB is binding as a single fusion protein or in a complex of initiation proteins. We analyzed the effect of removing Gal4 on the two fusions (DB-Spo11 and DBRec107) with the highest and lowest levels of DSBs. After removing Gal4 protein, DSBs at GAL2 were increased by 50% in each strain, and the relative difference between DB-SPO11 and DB-REC107 strains was not significantly different (P ¼ 0.52). Thus, variations in DSB frequency in strains with different fusion proteins are not easily explained by differential competition with Gal4 protein for binding to UASGAL sites. A third possibility is that the DSB frequency reflects a sequential assembly of subcomplexes. Three subcomplexes (the Spo11-subcomplex, the Rec114-subcomplex, and the MRX-subcomplex) exist among the initiation proteins (see Introduction). Our data indicate that the Spo11-subcomplex components are, on average, most effective at creating DSBs at GAL2 (average DSB value per protein ¼ 9.9%). The Rec114-subcomplex components (average DSB value per protein ¼ 4.0%) are generally less effective at creating DSBs. The MRXsubcomplex (as indicated only by Mre11) was ineffective
456
D. R. Koehn et al. TABLE 6 Quantification of DSB bands produced with DB-REC fusions in REC1 diploids
Relevant genotype DB-SPO11 DB-REC102 DB-REC104 DB-SKI8 DB-REC114 DB-REC107 DB-MEI4 DB-MRE11
REC1 diploid at GAL2: Avg. DSB (%) at 15 hra A 1 Bb,d
C 1 Db,d
7.3 8.7 4.8 2.2 6.3 1.3 3.2 0.4
1.8 3.8 1.5 1.7 2.4 1.6 1.1 NDf
Totalc,d
Change w.r.t.e recD
6 6 6 6 6 6 6 6
1.7 Y 1.7 [ 2.0 Y 1.1 Y 2.9 [ 1.0 — 1.5 Y NAg
9.1 12.4 6.2 3.9 8.7 2.8 4.2 0.4
1.5 1.4 0.8 1.4 0.9 0.9 1.1 0.2
Strains and plasmids are as in Figure 7. a Average of two independent experiments. b A 1 B and C 1 D are the sums of DSBs observed with bands A and B, C and D, respectively. c Total is the sum of DSBs at A, B, C, D and is the average of the number of experiments indicated 6 standard deviation. d All DSB values have been corrected for plasmid loss as described in materials and methods. e w.r.t., with respect to. f No detectable DSBs. g Not applicable because no DSBs above mitotic levels were observed.
at creating DSBs at the coldspot. Therefore, differences in the amount of DSBs could reflect the normal order of assembly; a fusion protein in a subcomplex that normally associates with DNA first would be better at attracting the other components. Of the four Spo11-subcomplex members, the tethered Ski8 protein is the least effective at inducing DSBs. Arora et al. (2004) showed that Ski8 associates with both chromatin loops and axes; in contrast, Rec102 and Spo11 were reported to be primarily on chromatin loops (Blat et al. 2002; Kee et al. 2004). It was proposed that Ski8 acts as a scaffold for the proper assembly of Spo11-Rec104-Rec102 (Arora et al. 2004; Li et al. 2006). If the main role of Ski8 were to assemble the other three components, then Ski8 may not be present at hotspots when DSBs are actually made. Perhaps the protein normally dissociates after bringing the other three together, and when continually present, as with DBSki8, actually reduces the efficiency of DSB formation. This view would predict that Rec103 should not be as effective in pulling down hotspots in ChIP experiments where DSB progression was blocked by rad50S. Double strand break pattern upstream of GAL2: Previous studies of tethered Spo11 showed only one DSB at GAL2 (Pecin ˜a et al. 2002). Our data indicate four DSBs. One possible explanation for the difference may be that Pecin ˜a et al. (2002) used strains of the SK1 background (Pecin ˜a et al. 2002), while we used the S288C background. It is possible that the GAL2 promoter regions of the two strains have sequence poly-
Figure 8.—Chromatin immunoprecipitation of DB-Mre11. Graph showing the ratio of GAL2 UASGAL (PP1, targeted hotspot) vs. CSF1 (PP2, coldspot) ChIP PCR product at 0, 4, 8, and 12 hr in sporulation (see materials and methods for details). Diamond bars represent cells with only vector and shaded bars represent cells with the DB-Mre11 fusion. Three independent trials were done and the values are mean 6 standard error.
morphisms, resulting in an altered pattern of breaks. Alternatively, there may be some technical difference in how we monitor DSBs. However, we observe the same four breaks (and the same patterns of breaks) when we probe the chromosome using the approach as Pecin ˜a et al. (2002) (Figure 4C). We detected doublet DSBs in the coding region of the ARE1 hotspot at the locations of two consensus UASGAL sites. Pecin ˜a et al. (2002) also observed that a DB-Spo11 fusion made breaks at these two sites. Gal4DB binds UASGAL as a dimer (Carey et al. 1989), allowing the initiation complex to potentially form on either side of it, which would result in doublets. This observation raises the possibility that the four DSB bands at GAL2 are actually two doublets produced from the fusions binding to two of the four UASGAL sites (Figure 4A). At this time we know of no reason why only two sites should be favored. The recombination initiation complex might be able to assemble in alternative ways: Meiotic recombination initiation requires the presence of multiple proteins; the simplest idea would be that initiation depends on these proteins interacting with a strictly defined spatial orientation. However, our results suggest that the idea of a complex, forming in a static, fixed way, is not sufficient. On average the Spo11-subcomplex members appear to recruit a functional complex most effectively, and the Rec114-subcomplex members cause DSBs at a
Meiotic Recombination Initiation
lower frequency. We propose that the functional initiation complex would form differently when different proteins are tethered. The result that seven different tethered proteins make breaks suggests there is flexibility in the ways the proteins assemble, all of which still place Spo11 in a proper position to create DSBs. Such flexibility in the assembly of a functional complex might be useful to ensure recombination initiation occurs if different proteins (or subcomplexes) interact with the DNA first; it would also explain, in part, why different experimental approaches have produced different conclusions about the order of assembly (see Introduction). We thank Jan Fassler, Tom Petes, Scott Keeney, and Karry Jannie for comments on the manuscript. We thank Kelley Foreman for laboratory assistance, Doug Houston for generously providing qPCR materials and expertise, Amnon Kohen for use of his Molecular Dynamics phosphorimager, and Josh Weiner for use of his Invitrogen magnetic separator. We also thank Eric Alani who provided a protocol for ChIP. D.R.K. was supported during summers by the Evelyn Hart-Watson and Avis Cone graduate fellowships and in the last stages of the work by National Science Foundation grant MCB-0743983. J.M.W. was supported by a University of Iowa summer honors undergraduate fellowship. The initial work was supported by National Science Foundation grant MCB-0083816. The majority of the work was supported by the Department of Biology (University of Iowa). The final experiments were supported by National Science Foundation grant MCB-0743983.
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