Selection of amino acid residues for targeted mutagenesis.
The overall sequence identity among Spo11p/Top6A family members is only 20 to 30% in pairwise combinations, but several blocks of much higher conservation are apparent (6
). These blocks correspond in many cases to core structural motifs identified in the Methanococcus jannaschii
Top6A crystal structure (32
). To identify residues in yeast Spo11p that might contribute directly to DNA cleavage, we first looked for conserved residues within the 5Y-CAP and Toprim domains whose side chains in M. jannaschii
Top6A lie near the catalytic tyrosine residue, near the acidic metal binding pocket or in the space between these putative components of the active site (Fig. ).We targeted the equivalent of eight such residues in S. cerevisiae
Spo11p for mutagenesis. We also chose three residues in Top6A whose side chains contribute prominently to the putative DNA binding surface, on the premise that these might be involved in DNA binding. These residues are not as highly conserved, but likely equivalent residues in Spo11p could be identified from sequence alignments and from homology-based structural modeling (Fig. and data not shown).
FIG. 1. (A) Orthogonal views of the M. jannaschii Top6A crystal structure, with B-form DNA (yellow) modeled into the putative DNA binding channel. The Top6A monomers are colored to indicate the 5Y-CAP (green) and Toprim (red) domains; the bound Mg2+ ions (more ...)
A total of 18 point mutations at 11 positions were generated and analyzed for their effect on Spo11p activity in vivo. The mutant proteins were tagged at their C termini with three repeats of the hemagglutinin (HA) epitope plus a hexahistidine sequence and were expressed under the control of the SPO11 promoter on an ARS/CEN vector in diploid spo11Δ/spo11Δ strains. Spo11p activity was assessed by measuring meiotic DSBs at the his4::LEU2 hotspot in both RAD50 and rad50S backgrounds, his4::LEU2 heteroallele recombination frequencies, and spore viability (an indirect measure of total recombination in the genome). DSB Southern blots for a representative subset of the mutants are presented in Fig. ,and the phenotypic analyses are summarized in Table . The spore viability and recombination frequency with a plasmid-borne wild-type SPO11 allele were slightly lower than with chromosome-borne alleles (compare Table with Table , below), most likely because plasmid loss renders a fraction of the cells spo11. For those cells that retained the plasmid, however, the copy number was at least two per cell on average, based on analysis of plasmid segregation in four-spore-viable tetrads (data not shown). For epitope-tagged proteins, all of the point mutants were expressed at levels comparable to wild-type levels, measured by Western blotting of whole-cell extracts (Fig. ). For the purpose of this analysis, we defined a residue as being essential for Spo11p function if mutation caused a ≥100-fold decrease in recombination frequency.
FIG. 2. (A) Effects of selected spo11 mutations on DSB formation. DSBs that were formed at the his4::LEU2 locus during meiosis in a spo11Δ rad50S strain (SKY103) carrying each of the indicated spo11-HA3His6 alleles on an ARS/CEN vector were analyzed by (more ...) Potential active site residues in the 5Y-CAP domain.
Tyr-135 is the only tyrosine absolutely conserved in all Spo11p/Top6A family members (Fig. ) and is thus thought to be the catalytic residue that carries out a nucleophilic attack on the DNA backbone, becoming covalently attached to the DNA. Consistent with this interpretation, Tyr-135 was previously shown to be essential for Spo11p activity in vivo (6
). We obtained similar results with an independently generated phenylalanine substitution at this position (12
; data not shown).
The arginine at position 131 is conserved in all Spo11p/Top6A family members (Fig. ), and an arginine is found at a similar position relative to the catalytic tyrosine in three-dimensional space in all 5Y-CAP topoisomerases (4
). Replacing it with alanine at this position severely compromised Spo11p activity in vivo (R131A
, Table ). Interestingly, a conservative substitution of lysine at this position (R131K
) supported high levels of meiotic intragenic recombination (44% of wild type) and spore viability (76% of wild type), but DSBs were more severely reduced: 5% of wild type in rad50S
(Table ) and <10% of wild type (i.e., undetectable) in RAD50
(data not shown). This apparent discrepancy between DSB levels and recombination frequencies appears to reflect effects of the mutation on the distribution of cleavage events at the recombination hotspot (see below).
Most Spo11p/Top6A family members have an acidic residue at the position equivalent to Asp-132 in Spo11p (Fig. ). Nevertheless, substituting alanine for Asp-132 had little, if any, effect on the ability of the mutant allele to complement the defects of a spo11 null (Table ), indicating that this residue is not critical for Spo11p catalytic activity in vivo. This result is consistent with the fact that this residue is not conserved in Neurospora crassa Spo11p (asparagine) or Drosophila melanogaster MeiW-68 (glycine).
Potential active site residues in the Toprim domain.
The three conserved acidic residues which coordinate a Mg2+ ion in the Top6A structure were each mutated to alanine or to glutamine or asparagine (E233A, E233Q, D288A, D288N, D290A, and D290N). Of these mutants, E233A, E233Q, D288A, and D288N were indistinguishable from a null mutation (Table ). In contrast, substitutions at Asp-290 had less effect: D290A showed 10 to 30% of wild-type levels for the parameters assayed, while D290N was nearly normal.
The position equivalent to Spo11p Glu-235 contains an acidic residue in nearly all of the eukaryotic Spo11p homologs but is not conserved in the archaebacterial Top6A proteins (Fig. ). Replacement with alanine at Glu-235 resulted in a relatively modest defect in intragenic recombination (35% of wild type) and spore viability (60% of wild type). However, as in the case of R131K (above), DSB frequency appeared to be more severely compromised (7% of wild type) and the distribution of cleavage sites at the his4::LEU2 hot spot was altered (see below).
The C-terminal regions of members of the Spo11p/Top6A family are poorly conserved, but one block of conserved sequence includes two residues (equivalent to Lys-384 and Glu-386 of S. cerevisiae Spo11p) that lie in or near the putative active site in M. jannaschii Top6A (Fig. ). Replacement by alanine at Glu-386 caused a modest defect (38 to 75% of wild type for the parameters assayed), whereas the K384A allele was severely compromised.
Mutations in residues potentially involved in binding DNA.
Mutants were also generated that affected each of three Spo11p residues equivalent to M. jannaschii Top6A residues which protrude into the putative DNA binding channel and which might be expected to interact directly with DNA. The first of these (equivalent to Gln-172 in yeast Spo11p) is glutamate in most of the archaeal proteins and serine, glycine, or alanine in most of the other eukaryotic proteins (Fig. ). Mutation of this residue in yeast Spo11p (Q172A and Q172R) had little if any effect on the parameters examined (Table ). The second such residue (equivalent to Phe-260) is a glutamine in most of the archaeal proteins but is a bulky hydrophobic residue in the eukaryotic proteins. Replacement by alanine at this position significantly decreased Spo11p activity in vivo (F260A). A charged amino acid substitution at this position supported normal amounts of recombination (F260R, Table ) but caused an alteration in DSB site specificity (see below). A third residue, Tyr-292, is more widely conserved between eukaryotes and archaebacteria; it is tyrosine, phenylalanine, or tryptophan in three-quarters of the members of the Spo11p/Top6A family and histidine in most of the remainder. Placing alanine at this position (Y292A) had little effect on Spo11p activity in vivo, but replacement with arginine (Y292R) inactivated Spo11p (Table ). A possible reason for this mutational effect is discussed below.
Synergistic defects from combining certain point mutations with the epitope tag.
The epitope-tagged version of Spo11p used for this analysis (Spo11-HA3His6p) supports high levels of recombination at 30°C. However, while this analysis was in progress, we discovered that the tag renders Spo11p slightly cold sensitive, indicating that the protein is not completely normal (21
). To address the concern that the phenotypes described above might be affected by the presence of the tag, a subset of the mutations was reengineered in an untagged form and analyzed as described above. For the E233A
mutations in the Toprim domain and for R131A
in the 5Y-CAP domain, the untagged mutant alleles were still severely compromised (Table ). These residues thus appear to be essential for Spo11p activity.
In contrast, a set of mutations that conferred a partial loss-of-function phenotype when epitope tagged (D290A, E235A, R131K, E386A, and F260A) supported significantly higher recombination levels when the tag was removed, in many cases indistinguishable from wild-type levels (Table ). The K384A mutant was even more striking: for this allele, intragenic recombination and spore viability were <1% of wild-type levels for the tagged form but were indistinguishable from wild-type levels for the untagged form. It thus appears that the D290A, E235A, R131K, K384A, E386A, and F260A mutations confer slight defects which are only revealed when Spo11p is sensitized by the modest defect caused by the C-terminal HA3His6 epitope tag.
Because the epitope tag caused a weak cold-sensitive phenotype by itself, we also asked whether conditional defects were associated with the point mutations that acted synergistically with the tag. None of the untagged point mutants was noticeably deficient relative to the wild type at elevated temperature (data not shown), but D290A, E235A, and F260A showed roughly 10-fold-lower intragenic recombination frequencies and spore viability at 16°C than at 30°C and K384A was reduced roughly three- to fourfold (Table ).
Alterations of DSB site preference.
A subset of the mutants that retained recombination-promoting activity (R131K
, and D290A
) generated noticeably unusual DSB patterns at site I of the his4
hotspot in Southern blots of agarose gels (Fig. and ; data not shown). In higher-resolution analysis on polyacrylamide gels, DSBs at site I were spread over a roughly 100- to 150-bp region in SPO11/SPO11 rad50S/rad50S
cells, as previously reported (41
) (Fig. ). At this resolution, the epitope tag had little if any effect on the distribution of DSBs (Fig. and data not shown), although the total DSB frequency was somewhat diminished, consistent with intragenic recombination frequencies measured with integrated constructs (see below). The four mutations that gave aberrant patterns on agarose gels gave even more dramatically altered patterns in this higher-resolution analysis (Fig. ). There were slight variations in DSB distribution (in addition to the previously established differences in DSB frequency; see above) between tagged and untagged alleles, but the major effects of these point mutations on DSB site preference appeared to be largely independent of the epitope tag. Untagged D290A
gave cleavage patterns similar to one another, whereas the patterns for R131K
were distinctly different.
FIG. 3. Alteration of the distribution of cleavage events at a recombination hotspot. kbp, kilobase pairs. ARS/CEN vectors carrying the indicated SPO11 alleles (with or without the HA3His6 tag, as indicated) were introduced into SKY103 (spo11Δ rad50S (more ...) Semidominance of DSB-defective alleles.
Two Spo11p monomers must act in concert to cleave both strands of the DNA duplex (15
). In the Top6A structure, the catalytic tyrosine of each monomer lies closer to the Toprim metal binding pocket of its dimer partner than it does to its own Toprim domain (Fig. ). This arrangement suggests that the dimer forms two hybrid active sites, each responsible for cleaving a single strand of the DNA duplex (32
). Extrapolating this dimeric structure to Spo11p predicts that catalytically inactive mutants should confer a semidominant-negative phenotype when coexpressed with the wild type, if Spo11p is limiting in vivo. To test this prediction, we replaced the normal SPO11
locus with each of several mutant alleles and tested whether single copies of the mutants were recessive, dominant, or semidominant relative to the wild type. We examined three severely compromised mutants (Y135F
, and D288N
) and one partial loss-of-function mutant (D290A
). Because of the effects of the epitope tag on Spo11p activity, the mutations were examined in four different configurations. Each configuration consisted of a test allele (wild-type or mutant) with or without the HA3His6 tag in combination with a wild-type SPO11
allele, also with or without the tag. All alleles were marked with the kanMX4
selectable marker inserted downstream of the SPO11
coding sequence. Intragenic recombination frequency at his4
and spore viability were assessed for each mutation in each configuration (Table ).
When neither allele was tagged (configuration A in Table ), the mutations had little or no effect on the parameters assayed. The spo11(Y135F)/SPO11 strain (SKY546) was indistinguishable from a matched hemizygous control (SKY486, SPO11/spo11Δ), with a modest decrease in the intragenic recombination frequency relative to a wild-type control and no significant change in spore viability (P > 0.1). Similar results were obtained with the E233A (SKY487) and D288N (SKY488) mutations. These results indicate that DSB-defective alleles of SPO11 are completely or nearly completely recessive when present in single copy along with a wild-type allele. The spo11(D290A)/SPO11 strain (SKY538, Table ) was essentially indistinguishable from the wild type, consistent with the fact that the untagged D290A allele supported nearly wild-type recombination levels on its own (Table ).
If the mutant and wild-type proteins can dimerize with one another, then the cell should contain roughly one-fourth the normal amount of wild-type Spo11p homodimer, with the remainder consisting of heterodimers and mutant homodimers, neither of which should be able to catalyze DSB formation. One explanation for the recessive nature of the DSB-defective alleles in configuration A would be that Spo11p activity is not limiting, such that a fourfold reduction in the amount of active protein has little effect on recombination frequencies. We therefore also tested the DSB-defective alleles under conditions in which Spo11p activity was slightly compromised, i.e., when the protein was tagged with an HA3His6 epitope tag (configuration B in Table ). Strikingly, the Y135F and D288N mutations were semidominant in this configuration, with intragenic recombination frequencies reduced to roughly 10 to 50% of the levels in matched homozygous and hemizygous controls and with spore viability reduced to roughly 50 to 60% of normal (SKY335 and SKY337, respectively). In contrast, the E233A mutation was at best only weakly semidominant: a strain heterozygous for this mutation (SKY587) had no detectable defect in recombination at his4::LEU2 but had a small but statistically significant decrease in spore viability (P < 0.01). The D290A mutation, which was partially defective for recombination initiation when tagged (see above), conferred an intermediate semidominant phenotype (SKY336, Table ). The decreased spore viability in these strains resulted mostly from an increase in the frequency of two- and zero-spore-viable tetrads, with little or no increase in one- and three-spore-viable tetrads, as expected for an increase in meiosis I nondisjunction caused by a recombination initiation defect (Fig. ).
FIG. 4. Spore viability patterns indicative of meiosis I nondisjunction in strains carrying semidominant configurations of SPO11 mutant alleles. Tetrads were dissected from strains with the indicated epitope-tagged alleles integrated at the SPO11 locus (configuration (more ...)
We also examined configurations in which only one of the SPO11 alleles was tagged. When the point mutant allele carried the tag, the mutation was recessive (configuration C, Table ). In contrast, if the mutant allele was untagged, the Y135F, E233A, and D288N mutations were dominant over a tagged wild-type allele (configuration D, Table ). Intragenic recombination frequency and spore viability were reduced to roughly 1 to 3% of the levels seen in a matched SPO11/spo11Δ control. The spo11(D290A)/spo11-HA3His6 heterozygote in configuration D was essentially normal, as expected because the untagged D290A mutant itself behaved normally.
A simple explanation for these dominance patterns would be that the tag causes a decrease in the steady-state level of the protein. Unfortunately, polyclonal anti-Spo11p antibodies that are sufficiently specific to detect the protein in crude lysates are presently unavailable to us, so it has not been possible to directly measure relative levels of tagged and untagged proteins. However, this hypothesis predicts that increasing the expression of a tagged allele should at least partially overcome its recessivity. We therefore introduced spo11-HA3His6 on a high-copy-number 2μm plasmid into SKY545 [spo11(Y135F)/spo11-HA3His6], resulting in a roughly 20- to 40-fold increase in the amount of tagged Spo11p. The increased Spo11-HA3His6p expression gave significant rescue of the recombination defect, with the recombination frequency at his4::LEU2 rising ca. 100-fold (Fig. ).
FIG. 5. Overexpression partially rescues the recessivity of spo11-HA3His6. Freq., frequency of; hr, hours. High-copy-number 2μm (2μ) vectors pDA7 (spo11-HA3His6) or pRS426 (vector control) were introduced into strain SKY545 [heterozygous for untagged (more ...)