Saccharomyces cerevisiae meiosis-specific hop1 protein and its putative zinc-finger motif promote synapsis between double-stranded DNA helices : a role for guanine quartets in ininterstitial pairing

Abstract

Meiosis is an evolutionarily conserved biological process that allows genetic exchange between paternal and maternal genomes in all sexually reproducing species. The hallmark features of meiosis include pairing of homologous chromosomes, recombination, and subsequent reductional division, resulting in the generation of haploid gametes. The successful outcome of this process relies on several key events occurring during meiosis I, one of which is the pairing of homologous chromosomes. In most eukaryotes, the pairing of homologs is facilitated by a meiosis-specific cytological structure, the synaptonemal complex (SC). SCs are proteinaceous zipper-like structures assembled along the paired homologs during the prophase of the first meiotic division. Apart from its role in chromosome pairing, SC has been shown to play a crucial role in the regulation of crossing over during meiotic recombination. At the ultrastructural level, SC is visualized as a tripartite structure consisting of a pair of lateral elements, a central element, and several interconnecting transverse elements. Although SCs were first discovered over five decades ago in insects, and the components identified from a number of species, its role in pairing of meiotic chromosomes and the molecular functions of its components has remained unclear.

Genetic analyses of meiotic chromosome synapsis and recombination in Saccharomyces cerevisiae has led to the identification of a number of genes that are essential for the proper assembly of SC. The meiosis-specific HOP1 (for Homolog Pairing) has been shown to encode a component of SC. hop1 diploids display reduced DSB levels as well as reduced inter-chromosomal recombination (<10%), less heteroduplex DNA formation, incomplete synapsis, and failure to produce viable spores. Immunochemical methods have localized the 70 kDa product of HOP1 to the lateral elements of SC. The deduced amino acid sequence of HOP1 revealed the presence of a putative zinc finger motif (residues 348–374 in the full-length protein), which is reminiscent of the Cys2/Cys2 type of zinc finger motif identified in several transcription factors. The 348CX2CX19CX2C374 motif of Hop1 appears to be crucial for its function in meiosis, since mutation of the conserved Cys (at position 371) to Ser in this motif resulted in defective sporulation and meiosis.

To understand the molecular function of HOP1 in chromosome synapsis, Hop1 protein was purified from mitotically dividing yeast cells harboring a recombinant HOP1 construct. Subsequently, functional characterization of purified Hop1 protein revealed it to be an oligomeric, structure-specific, DNA-binding protein. Hop1 bound duplex DNA at a stoichiometric ratio of 50 bp/monomer and exhibited high affinity for G-rich oligonucleotides. Most intriguingly, Hop1 displayed higher affinity for G4 DNA (a configuration arising from stacks of guanine quartets, in which four guanines are hydrogen-bonded in a cyclic planar array) as well as favored its formation from unfolded oligonucleotides containing arrays of guanine residues.

While the correlation between high-affinity binding and formation of G4 DNA by Hop1 protein strongly implicates a possible role for this structure in pairing of meiotic chromosomes, it is unknown whether Hop1 plays a role in bringing two duplex DNA molecules together during meiosis. In this study, we have examined the ability of Hop1 to promote pairing of double-stranded DNA helices and the molecular functions of Hop1’s putative zinc finger. A 48 bp duplex DNA containing a central stretch of eight G/C bp was used in this study. Consistent with our previous observations, Hop1 was proficient in binding to the 48 bp duplex DNA containing 8 bp G/C array. To explore whether Hop1 can align two DNA double helices in juxtaposition, Hop1-DNA complexes were treated with proteinase K and SDS and analyzed by native PAGE followed by autoradiography. Interestingly, it was observed that a small proportion of the 48 bp duplex DNA was converted into a lower mobility product corresponding to the size of a synapsed dimer (~150 bp) of 48 bp duplex DNA molecules (referred to as synapsis product). The anomalous mobility of the dimer, the blunt-ended nature of the duplex DNA used, and the absence of ligase in the Hop1 protein preparation indicated that the synapsis product is not a tandem dimer but a side-by-side dimer of two DNA double helices.

Although these results revealed the ability of Hop1 protein to promote synapsis of DNA double helices, the extent of synapsis was poor. To distinguish between the possibility of whether the poorer synapsis is an inherent property of Hop1, or a limitation due to its inability to unwind duplex DNA, a 48 bp duplex DNA was designed with eight consecutive mismatched G/G base pairs embedded centrally in Watson-Crick duplex context (although it is not a natural substrate in vivo). Hop1 formed a complex with the duplex DNA containing a (G/G)8 stretch. To examine if Hop1 could mediate the synapsis between such duplex DNA, the Hop1-DNA complexes were deproteinized and analyzed as described above. Intriguingly, Hop1 was more efficient in promoting the synapsis of 48 bp mismatched duplex DNA compared to that of a DNA duplex containing a (G/C)8 sequence.

Subsequent experiments carried out with the mismatched duplex DNA revealed that synapsis promoted by Hop1 is rapid (occurring within 2 min of incubation) compared to self-assembly (mediated by salt), saturable, and sensitive to the presence of unlabeled homologous competitor DNA. Interstitial pairing was Hop1-specific, since RecA (a strand exchange protein) or histone H1 (a basic protein) failed to promote synapsis between 48 bp double helical DNA with a (G/G)8 stretch. Insights into the mechanistics of synapsis from restriction cleavage sensitivity assay and synapsis between heterologous duplex DNA implicated a role of G/G bp in synapsis, and excluded the involvement of arms flanking the central (G/G)8 region. The role of G-residues in the G/G region in Hop1-mediated synapsis was underscored by the results from mutant substrates containing varying numbers of G/G bp, which showed that Hop1 required a minimum of four contiguous G/G base pairs for synapsis. Furthermore, results from truncated duplex DNA substrates suggested that the minimum length required for Hop1-mediated synapsis is in the range of 38–48 bp, which is reminiscent of the minimum length requirement for DNA binding of Hop1.

The formation of G-quartets is best characterized with methylation protection/interference assays, wherein the involvement of N7 of guanine in Hoogsteen base pairing renders them less accessible to methylation by dimethyl sulfate (which predominantly methylates the N7 position of guanine and N3 of adenine). Accordingly, methylation interference assays showed significant protection of guanine residues in the (G/G)8 region in the synapsis product in comparison with that of duplex DNA, suggesting that interstitial pairing of duplex DNA involves the formation of guanine quartets. These studies demonstrate that Hop1 protein promotes synapsis of DNA double helices via the formation of G-quartets, which is independent of homology in the flanking sequences.

To understand the biochemical functions of the putative zinc finger motif of Hop1, we generated two peptides of 36 amino acid residues each, corresponding to the zinc finger motif sequence (wild type) and its mutant with a substitution at C371S (mutant peptide). The ability of the Hop1 zinc finger peptides to interact with zinc, a feature intrinsic to canonical zinc finger motifs, was assessed by radioactive zinc-binding assays. The wild type peptide displayed high affinity for zinc while the mutant peptide showed markedly reduced zinc-binding activity. Competitive metal-binding assays revealed that the wild type peptide binds zinc in a tetrahedral coordination, a characteristic feature of zinc finger motifs. In addition, circular dichroism (CD) measurements revealed that the wild type Hop1 zinc finger peptide undergoes conformational changes upon interaction with zinc, wherein the random coil/-sheet is converted into an ordered -helical conformation. The affinity of the wild type peptide towards zinc was quantitated using the changes in negative ellipticity at 222 nm, which yielded a Kd of 2.5 × 10^5 M. Together, these results indicate that the Hop1 zinc finger peptide functions as an independent metal-binding module, a characteristic feature of zinc fingers.

To explore the role of the Hop1 putative zinc finger motif in interaction with DNA, the wild type Hop1 zinc finger peptide or its C371S mutant was incubated with various topological forms of M13 DNA and analyzed by electrophoretic mobility shift assays. Significantly, the wild type peptide was proficient in interacting with all the topological forms of M13 DNA (supercoiled, linear, and single-stranded), while the mutant peptide failed to bind DNA. These results suggest that the putative zinc finger motif of Hop1 is involved in DNA binding, a feature universal to most zinc finger motifs.

To address the sequence specificity of DNA binding exhibited by the wild type peptide, we assayed its interaction with oligonucleotides containing stretches of guanine residues by mobility shift assays. The putative zinc finger motif, but not its C371S mutant, exhibited high affinity of interaction with the G-rich oligonucleotides, in a manner similar to the full-length Hop1. Consequently, we examined the ability of the zinc finger motif to bind G4 DNA. Interestingly, the wild type peptide, but not its mutant, exhibited high affinity for G4 DNA as well. Nitrocellulose filter-binding assays were performed to determine the relative affinity of the Hop1 zinc finger peptide towards various DNA substrates. Comparison of the Kd values revealed that the wild type peptide binds G4 DNA with higher affinity (0.2 M), which is about 15-fold greater than that for normal duplex DNA (3 M). It is thus interesting to note that the Hop1 zinc finger motif, like the full-length Hop1, is capable of distinguishing B-form DNA from G4 DNA. The peptide-DNA interactions were ascertained by circular dichroism measurements, which suggested DNA-induced conformational changes in the wild type but not the mutant peptide.

Thus, DNA-binding assays indicate that the putative zinc-finger motif of Hop1 is indeed a functional zinc finger, which exhibits features reminiscent of the full-length Hop1. To gain further insights into the biochemical functions of the zinc finger motif, its ability to promote the formation of G4 DNA / synapsis of duplex DNA were explored using assays as described for Hop1 protein. Significantly, the wild type zinc finger peptide was proficient in promoting the formation of G4 DNA from monomeric G-rich substrates as well as synapsis of double helical DNA containing a (G/G)8 stretch. The synapsis mediated by the wild type peptide was saturable and dependent on an array of four contiguous G/G stretches. The conserved Cys residue in the putative zinc finger motif of Hop1 was necessary and sufficient for the display of these activities. Unlike the full-length Hop1, the wild type zinc finger motif could mediate synapsis between 28 bp (G/G)8 mismatched duplex DNA. The involvement of guanine quartets in zinc finger-mediated synapsis was established by methylation interference assays.

These observations suggest that the Hop1 zinc finger motif is sufficient for the display of all of the known activities of full-length Hop1 under in vitro conditions. Structural modeling of the Hop1 zinc finger motif revealed that it is an atypical zinc finger motif wherein the positions of the -helix and the -sheets are inverted to give rise to an motif rather than the motif present in TFIIIA-type zinc fingers.

In summary, the present work implicates a direct role for Hop1 and its zinc finger motif in meiotic chromosome pairing owing to their intrinsic ability to promote interstitial pairing of double-stranded DNA helices via G4 DNA formation. To our knowledge, Hop1 is the first SC component whose biochemical function has been understood. This study forms the first report of a protein that can mediate synapsis of double helical DNA.