付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23114号 / 医科博第125号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 篠原 隆司, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20212号 / 理博第4297号 / 新制||理||1617(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 准教授 小山 時隆, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

Yes / Abstract The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1-derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. 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 VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions.

Homologous recombination (HR) is essential for the repair of DNA doublestrand breaks (DSBs) and damaged replication forks. However, HR can also cause gross chromosomal rearrangements (GCRs) by producing crossovers (COs), resulting in the reciprocal exchange of sequences between non-sister chromatids. Therefore, HR-mediated GCRs are suppressed via the promotion of HR pathways that favour noncrossover (NCO) formation, such as the synthesis-dependent strand annealing (SDSA) and dissolution pathways, which are modulated by Mph1 and Sgs1 helicases, respectively. The mismatch repair (MMR) pathway is intricately associated with HR via its roles in repairing mismatches on heteroduplex DNA that can arise during HR and in preventing homeologous recombination. Using a plasmid break-repair assay, we have revealed a novel, MMR-independent role of MutSα in promoting the formation of a subset of COs that is specifically supressible by Mph1, during HR between two completely homologous sequences. In contrast, the MMR-dependent function of MutSα, together with Mph1 and Sgs1, was shown to be required for the suppression of CO formation during homeologous recombination. These data indicate that Mph1 can both antagonise and promote the functions of MutSα during DSB repair, depending on the levels of homology between the two recombining sequences. COs are generated by the resolution of Holliday junction (HJ) intermediates formed at the terminal stages of HR. Several S.cerevisiae proteins such as Yen1, Mus81, Slx1 and Rad1 have been implicated in HJ resolution. However, the in vivo roles of these proteins in HJ resolution remain to be confirmed. To directly and quantitatively monitor in vivo HJ resolution in S.cerevisiae, a transformation-based HJ resolution assay using a plasmid-borne HJ substrate has been developed. Using this system, we have demonstrated an in vivo HJ resolution function of Yen1, which acts redundantly with Mus81. Moreover, these redundant activities of Yen1 and Mus81 are essential for survival during replication stress, but are dispensable for DSB repair. An Slx4 and Rad1-dependent in vivo HJ resolution activity was also observed in the absence of Yen1 and Mus81 that was suppressed by presence of Slx1. Models describing how the nucleases interact to process HJs in vivo will be discussed.

Meiosis is a specialized type of cell division where two rounds of chromosome segregation follow a single round of DNA duplication resulting in the formation of four haploid daughter cells. Once the DNA replication is complete, the homologous chromosomes pair and recombine during the meiotic prophase I, giving rise to genetic diversity in the gametes. The process of homology search during meiosis is broadly divided into recombination-dependent (involves the formation of double-strand breaks) and recombination-independent mechanisms. In most eukaryotic organisms, pairing of homologs, recombination and chromosome segregation occurs in the context of a meiosis-specific proteinaceous structure, known as the synaptonemal complex (SC). The electron microscopic visualization of SC has revealed that the structure is tripartite with an electron-dense central element and two lateral elements that run longitudinally along the entire length of paired chromosomes. Transverse filaments are protein structures that connect the central region to the lateral elements. Genetic analyses in budding yeast indicate that mutations in SC components or defects in SC formation are associated with chromosome missegregation, aneuploidy and spore inviability. In humans, defects in SC assembly are linked to miscarriages, birth defects such as Down syndrome and development of certain types of cancer. In Saccharomyces cerevisiae, genetic screens have identified several mutants that exhibit defects in SC formation culminate in a decrease in the frequency of meiotic recombination, spore viability and improper chromosome segregation. Ten meiosis-specific proteins, viz. Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8, have been shown to be the bona fide components of SC and/or associated with SC function. S. cerevisiae HOP1 (HOmolog Pairing) gene was isolated in a genetic screen for mutants that showed defects in homolog pairing and, consequently, reduced levels of interhomolog recombination (10% of wild-type). Amino acid sequence alignment together with genetic and biochemical analyses revealed that Hop1 is a 70 kDa protein with a centrally embedded essential zinc-finger motif (Cys2/Cys2) and functions in polymeric form. Previous biochemical studies have also shown that Hop1 is a structure-specific DNA binding protein, which exhibits high affinity for the Holliday junction (HJ) suggesting a role of this protein in branch migration of the HJ. Furthermore, Hop1 displays high affinity for G-quadruplex structures (herein after referred to as GQ) and also promotes the formation of GQ from unfolded G-rich oligonucleotides. Strikingly, Hop1 promotes pairing between two double-stranded DNA molecules via G/C-rich sequence as well as intra- and inter-molecular pairing of duplex DNA molecules. Structure-function analysis suggested that Hop1 has a modular organization consisting of a protease-sensitive N-terminal, HORMA domain (characterized in Hop1, Rev7, Mad2 proteins) and protease-resistant C-terminal domain, called Hop1CTD. Advances in the field of DNA quadruplex structures suggest a significant role for these structures in a variety of biological functions such as signal transduction, DNA replication, recombination, gene expression, sister chromatid alignment etc. GQs and i-motif structures that arise within the G/C-rich regions of the genome of different organisms have been extensively characterized using biophysical, biochemical and cell biological approaches. Emerging studies with guanine- and cytosine-rich sequences of several promoters, telomeres and centromeres have revealed the formation of GQs and i-motif, respectively. Although the presence of GQs within cells has been demonstrated using G4-specific antibodies, in general, the in vivo existence of DNA quadruplex structures is the subject of an ongoing debate. However, the identification and isolation of proteins that bind and process these structures support the idea of their in vivo existence. In S. cerevisiae, genome-wide survey to identify conserved GQs has revealed the presence of ~1400 GQ forming sequences. Additionally, these potential GQ forming motifs were found in close proximity to promoters, rDNA and mitosis- and meiosis-specific double-strand break sites (DSBs). Meiotic recombination in S. cerevisiae as well as humans occurs at meiosis-specific double-strand break (DSBs) sites that are embedded within the G/C-rich sequences. However, much less is known about the structural features and functional significance of DNA quadruplex motifs in sister chromatid alignment N during meiosis. Therefore, one of the aims of the studies described in this thesis was to investigate the relationship between the G/C-rich motif at a meiosis-specific DSB site in S. cerevisiae and its ability to form GQ and i-motif structures. To test this hypothesis, we chose a G/C-rich motif at a meiosis-specific DSB site located between co-ordinates 1242526 to 1242550 on chromosome IV of S. cerevisiae. Using multiple techniques such as native gel electrophoresis, circular dichroism spectroscopy, 2D NMR and chemical foot printing, we show that G-rich motif derived from the meiosis-specific DSB folds into an intramolecular GQ and the complementary C-rich sequence folds into an intramolecular i-motif, the latter under acidic conditions. Interestingly, we found that the C-rich strand folds into i-motif at near neutral pH in the presence of cell-mimicking molecular crowding agents. The NMR data, consistent with our biochemical and biophysical analyses, confirmed the formation of a stable i-motif structure. To further elucidate the impact of these quadruplex structures on DNA replication in vitro, we carried out DNA polymerase stop assay with a template DNA containing either the G-rich or the C-rich sequence. Primer extension assays carried out with Taq polymerase and G-rich template blocked the polymerase at a site that corresponded to the formation of an intramolecular GQ. Likewise, primer extension reactions carried out with KOD-Plus DNA polymerase and C-rich template led to the generation of a stop-product at the site of the formation of intramolecular I -motif under acidic conditions (pH 4.5 and pH 5.5). However, polymerase stop assay performed in the presence of single-walled carbon nanotubes (SWNTs) that stabilize I -motif at physiological pH blocked the polymerase at the site of intramolecular I -motif formation, indicating the possible existence of i-motif in the cellular context. Taken together, these results revealed that the G/C-rich motif at the meiosis-specific DSB site folds into GQ and i-motif structures in vitro. Our in vitro analyses were in line with our in vivo analysis that examined the ability of the G/C-rich motif to fold into quadruplex structures in S. cerevisiae cells. Qualitative microscopic analysis and quantitative analysis with plasmid constructs that harbour the GQ or i-motif forming sequence revealed a significant decrease in the GFP expression levels in comparison to the control. More importantly, all the assays performed with the corresponding mutant sequences under identical experimental conditions did not yield any quadruplex structures, suggesting the involvement of contagious guanine and cytosine residues in the structure formation. Prompted by our earlier results that revealed high binding affinity of Hop1 for GQ, we wished to understand the role of the GQ and i-motif structures during meiosis by analysing their interaction with Hop1 and its truncated variants (HORMA and Hop1CTD). In agreement with our previous observations, Hop1 and Hop1CTD associated preferentially with GQ DNA. Interestingly, whereas the full-length Hop1 showed much weaker binding affinity for i-motif DNA, Hop1 C-terminal fragment but not its N-terminal fragment exhibited robust i-motif DNA binding activity. We have previously demonstrated that Hop1 promotes intermolecular synapsis between synthetic duplex DNA molecules containing a G/C-rich sequence. Hence, to understand the functional role of the quadruplex structures formed at the meiosis-specific G/C-rich motif, we examined the ability of Hop1 to promote pairing between linear duplex DNA helices containing the G/C-rich motif. DNA pairing assay indicated that binding of Hop1 to the G/C-rich duplex DNA resulted in the formation of a side-by-side synapsis product. Under similar conditions, Hop1 was unable to pair mutant duplex DNA molecules suggesting the involvement of the G/C-rich motif in the formation of the synapsis product. Our results were substantiated by the observation that yeast Rad17 failed to promote pairing between duplex DNA molecules with a centrally embedded G/C-rich motif. Altogether, these results provide important structural and functional insights into the role of quadruplex structures in meiotic pairing of homologous chromosomes. The second part of the thesis focuses on the biochemical and functional properties of Red1 protein, a component of S. cerevisiae lateral element. RED1 was identified in a screen for meiotic lethal, sporulation proficient mutants. Genetic, biochemical and microscopic analyses have demonstrated the physical interaction between Hop1 and Red1. Given this, hop1 and red1 mutants display similar phenotypes such as chromosome missegregation and spore inviability and thus are placed under the same epistasis group. However, unlike hop1 mutants, red1 mutants show complete absence of SC. RED1 overexpression suppressed certain non-null hop1 phenotypes, indicating that these proteins may have partially overlapping functions. Further, although the functional significance is unknown, chromatin immunoprecipitation studies have revealed the localization of Red1 to the GC-rich regions (R-bands) in the genome, considered to be meiotic recombination hotspots. Although the aforementioned genetic studies suggest an important role for Red1 in meiosis, the exact molecular function of Red1 in meiotic recombination remains to be elucidated. To explore the biochemical properties of Red1, we isolated the S. cerevisiae RED1 gene, cloned, overexpressed, and purified the protein to near homogeneity. Immunoprecipitation assays using meiotic cells extracts suggested that Red1 exists as a Homodimer linked by disulphide-bonds under physiological conditions. We characterized the DNA binding properties of Red1 by analysing its interaction with recombination intermediates that are likely to form during meiotic recombination. Protein-DNA interaction assays revealed that Red1 exhibits binding preference for the Holliday junction over replication fork and other recombination intermediates. Notably, Red1 displayed ~40-fold higher binding affinity for GQ in comparison with HJ. The observation that Red1 binds robustly to GQs prompted us to examine if Red1 could promote pairing between duplex DNA helices with the G/C-rich sequences similar to Hop1. Interestingly, we found that Red1 failed to promote pairing between dsDNA molecules but potentiated Hop1 mediated pairing between duplex DNA molecules. Our AFM studies with linear and circular DNA molecules along with Red1 suggested a possible role of Red1 in DNA condensation, bridging and pairing of double-stranded DNA helices. Bioinformatics analysis of Red1 indicated the lack of sequence or structural similarity to any of the known proteins. To elucidate structure-function relationship of Red1, we generated several N- and C-terminal Red1 truncations and studied their DNA binding properties. Our results indicated that the N-terminal region comprising of 678 amino acid residues constitutes the DNA-binding region of Red1. The N-terminal region, called RNTF-II, displayed similar substrate specificity comparable to that of full-length Red1. Interestingly, site-directed mutagenesis studies with the Red1 C-terminal region revealed the involvement of two cysteine residues at position 704 and 707 in the disulfide bond mediated intermolecular dimer formation. Finally, to understand the functional significance of Red1 truncations we analyzed the subcellular localization of Red1 and its truncations. We made translation fusions of RED1 and its truncations by placing their corresponding nucleotide sequences downstream of GFP coding sequence in yeast expression vector. Confocal microscopy studies with S. cerevisiae cells transformed with the individual plasmid constructs indicated that the N-terminal variants localized to the nucleus, whereas the C-terminal variants did not localize to the nucleus. These results suggest that NLS-like motifs are embedded in the N-terminal region of the protein. Furthermore, other results indicated that the N-terminal region contains functions such as DNA-binding and intermolecular bridging of non-contiguous DNA segments. Altogether, these findings, on the one hand, provide insights into the molecular mechanism underlying the functions of Hop1 and Red1 proteins and, on the other, support a role for DNA quadruplex structures in meiotic chromosome synapsis and recombination.

Mon projet de recherche avait pour but de caractériser le rôle de deux protéines, ArgR et PepA, qui agissent en tant que facteurs accessoires de la recombinaison au niveau de deux sites cer du plasmide ColE1 présent dans la bactérie Escherichia coli. Ces deux protéines, couplées aux deux recombinases à tyrosine XerC et XerD, permettent la catalyse de la recombinaison site spécifique au niveau de la séquence cer, convertissant les multimères instables de ColE1 en monomères stables. Cette étude a principalement porté sur la région C-terminale de la protéine ArgR. Cette région de la protéine ArgR possède une séquence en acides-aminés et une structure similaire à celle de la protéine AhrC de Bacillus subtilis. De plus, AhrC, le répresseur de l’arginine de cette bactérie, est capable de complémenter des Escherichia coli mutantes déficientes en ArgR. Les régions C-terminales de ces protéines, montrent une forte similarité. De précédents travaux dans notre laboratoire ont démontré que des mutants d’ArgR comprenant des mutations dans cette région, en particulier les mutants ArgR149, une version tronquée d’ArgR de 149 acides-aminés, et ArgR5aa, une version comprenant une insertion de cinq acides-aminés dans la partie C-terminale, perdaient la capacité de permettre la recombinaison au niveau de deux sites cer présents dans le plasmide pCS210. Malgré cette incapacité à promouvoir la réaction de recombinaison en cer, ces deux mutants étaient toujours capables de se lier spécifiquement à l’ADN et de réprimer une fusion argA :: lacZ. Dans ce travail, les versions mutantes et sauvages d’ArgR furent surexprimées en tant que protéines de fusion 6-histidine. Des analyses crosslinking ont montré que la version sauvage et ArgR5aa pouvaient former des hexamères in-vitro de manière efficace, alors qu’ArgR149 formait des multimères de plus faible poids moléculaire. Des formes tronquées d’ArgR qui comportaient 150 acides-aminés ou plus, étaient encore capables de permettre la recombinaison en cer. Les mutants par substitution ArgRL149A et ArgRL151A ont tous montré que les substitutions d’un seul acide-aminé au sein de cette région avaient peu d’effets sur la recombinaison en cer. Les expériences de crosslinking protéine-à-protéine ont montré que le type sauvage et les formes mutantes d’ArgR étaient capables d’interagir avec la protéine accessoire PepA, également impliquée dans la recombinaison en cer. Les expériences de recombinaison in-vitro utilisant la forme sauvage et les formes mutantes d’ArgR combinées avec les protéines PepA, XerC et XerD purifiées, ont montré que le mutant ArgR149 ne soutenait pas la recombinaison, mais que le mutant ArgR5aa permettait la formation d’une jonction d’Holliday. Des expériences de topologie ont montré que PepA était capable de protéger l’ADN de la topoisomérase 1, et d’empêcher ArgRWt de se lier à l’ADN. Les deux mutants ArgR149 et ArgR5aa protègent aussi l’ADN avec plus de surenroulements. Quand on ajoute PepA, les profils de migration montrent un problème de liaison des deux mutants avec PepA. D’autres expériences impliquant le triplet LEL (leucine-acide glutamique-leucine) et les acides-aminés alentour devraient être réalisés dans le but de connaitre l’existence d’un site de liaison potentiel pour PepA. / My research project involved the role of two proteins, ArgR and PepA, which act as accessory factors in the ColE1 cer recombination system from the gram negative bacteria Escherichia coli. These two proteins, in addition to the tyrosine recombinases XerC and XerD, catalyze a site-specific recombination event at the cer sequence which converts unstable multimeric forms of ColE1 into more stable monomers. Our study mainly focused on the C-terminal end of the ArgR. This region of the ArgR protein possesses a structural and amino acid sequence similarity with the AhrC protein from Bacillus subtilis. Moreover, AhrC, the Arginine repressor of this bacterium, is able to complement Escherichia coli mutants deficient in ArgR. The C-terminal regions of these proteins, display a very high region of similarity. Previous work from our laboratory has shown that ArgR mutants with mutations in this region, especially the mutants ArgR149, a truncated 149 amino acids form of ArgR, and ArgR5aa, a form containing a five amino acid insertion in the C-terminal part, lost the ability to perform a recombination reaction at two cer sites in the plasmid pCS210. Despite this defect in promoting cer recombination, the mutants were still able to bind specifically to DNA, and to repress an argA :: lacZ genetic fusion. In this work, both wild type and mutant ArgR proteins were overexpressed as 6-histidine fusion proteins. Crosslinking analysis showed that both wild type and ArgR5aa efficiently formed hexamers in vitro, while ArgR149 formed lower molecular weight multimers. Truncated forms of ArgR that were 150 amino acids or longer, were able to support cer recombination. The substitution mutants between positions 149 to 151 all showed that single amino acid substitutions at this region had little effect on cer recombination. Protein-protein crosslinking experiments showed that wild type and mutant forms of ArgR, were able to interact with and the other accessory protein involved in cer recombination, PepA. In vitro recombination experiments using wild type and mutant forms of ArgR, combined with purified PepA, XerC and XerD showed that the ArgR149 mutant did not support recombination, but the ArgR5aa mutant did promote Holliday junction formation, raising the possibility that these two mutants interact differently with the Xer recombination machinery. Topology experiments showed that after adding topoisomerase 1, PepA is able to protect DNA from topoisomerase 1, and prevent ArgRWt binding to DNA. The two mutants ArgR149 and ArgR5aa are protecting DNA with more supercoiling. When PepA is added, migration profiles with the two mutants showed a binding problem with PepA. Other experiments involving the LEL triplet (leucine-glutamic acid-leucine) and amino-acids around it should be done in order to know the existence of a possible binding site for PepA.

Mon projet de recherche avait pour but de caractériser le rôle de deux protéines, ArgR et PepA, qui agissent en tant que facteurs accessoires de la recombinaison au niveau de deux sites cer du plasmide ColE1 présent dans la bactérie Escherichia coli. Ces deux protéines, couplées aux deux recombinases à tyrosine XerC et XerD, permettent la catalyse de la recombinaison site spécifique au niveau de la séquence cer, convertissant les multimères instables de ColE1 en monomères stables. Cette étude a principalement porté sur la région C-terminale de la protéine ArgR. Cette région de la protéine ArgR possède une séquence en acides-aminés et une structure similaire à celle de la protéine AhrC de Bacillus subtilis. De plus, AhrC, le répresseur de l’arginine de cette bactérie, est capable de complémenter des Escherichia coli mutantes déficientes en ArgR. Les régions C-terminales de ces protéines, montrent une forte similarité. De précédents travaux dans notre laboratoire ont démontré que des mutants d’ArgR comprenant des mutations dans cette région, en particulier les mutants ArgR149, une version tronquée d’ArgR de 149 acides-aminés, et ArgR5aa, une version comprenant une insertion de cinq acides-aminés dans la partie C-terminale, perdaient la capacité de permettre la recombinaison au niveau de deux sites cer présents dans le plasmide pCS210. Malgré cette incapacité à promouvoir la réaction de recombinaison en cer, ces deux mutants étaient toujours capables de se lier spécifiquement à l’ADN et de réprimer une fusion argA :: lacZ. Dans ce travail, les versions mutantes et sauvages d’ArgR furent surexprimées en tant que protéines de fusion 6-histidine. Des analyses crosslinking ont montré que la version sauvage et ArgR5aa pouvaient former des hexamères in-vitro de manière efficace, alors qu’ArgR149 formait des multimères de plus faible poids moléculaire. Des formes tronquées d’ArgR qui comportaient 150 acides-aminés ou plus, étaient encore capables de permettre la recombinaison en cer. Les mutants par substitution ArgRL149A et ArgRL151A ont tous montré que les substitutions d’un seul acide-aminé au sein de cette région avaient peu d’effets sur la recombinaison en cer. Les expériences de crosslinking protéine-à-protéine ont montré que le type sauvage et les formes mutantes d’ArgR étaient capables d’interagir avec la protéine accessoire PepA, également impliquée dans la recombinaison en cer. Les expériences de recombinaison in-vitro utilisant la forme sauvage et les formes mutantes d’ArgR combinées avec les protéines PepA, XerC et XerD purifiées, ont montré que le mutant ArgR149 ne soutenait pas la recombinaison, mais que le mutant ArgR5aa permettait la formation d’une jonction d’Holliday. Des expériences de topologie ont montré que PepA était capable de protéger l’ADN de la topoisomérase 1, et d’empêcher ArgRWt de se lier à l’ADN. Les deux mutants ArgR149 et ArgR5aa protègent aussi l’ADN avec plus de surenroulements. Quand on ajoute PepA, les profils de migration montrent un problème de liaison des deux mutants avec PepA. D’autres expériences impliquant le triplet LEL (leucine-acide glutamique-leucine) et les acides-aminés alentour devraient être réalisés dans le but de connaitre l’existence d’un site de liaison potentiel pour PepA. / My research project involved the role of two proteins, ArgR and PepA, which act as accessory factors in the ColE1 cer recombination system from the gram negative bacteria Escherichia coli. These two proteins, in addition to the tyrosine recombinases XerC and XerD, catalyze a site-specific recombination event at the cer sequence which converts unstable multimeric forms of ColE1 into more stable monomers. Our study mainly focused on the C-terminal end of the ArgR. This region of the ArgR protein possesses a structural and amino acid sequence similarity with the AhrC protein from Bacillus subtilis. Moreover, AhrC, the Arginine repressor of this bacterium, is able to complement Escherichia coli mutants deficient in ArgR. The C-terminal regions of these proteins, display a very high region of similarity. Previous work from our laboratory has shown that ArgR mutants with mutations in this region, especially the mutants ArgR149, a truncated 149 amino acids form of ArgR, and ArgR5aa, a form containing a five amino acid insertion in the C-terminal part, lost the ability to perform a recombination reaction at two cer sites in the plasmid pCS210. Despite this defect in promoting cer recombination, the mutants were still able to bind specifically to DNA, and to repress an argA :: lacZ genetic fusion. In this work, both wild type and mutant ArgR proteins were overexpressed as 6-histidine fusion proteins. Crosslinking analysis showed that both wild type and ArgR5aa efficiently formed hexamers in vitro, while ArgR149 formed lower molecular weight multimers. Truncated forms of ArgR that were 150 amino acids or longer, were able to support cer recombination. The substitution mutants between positions 149 to 151 all showed that single amino acid substitutions at this region had little effect on cer recombination. Protein-protein crosslinking experiments showed that wild type and mutant forms of ArgR, were able to interact with and the other accessory protein involved in cer recombination, PepA. In vitro recombination experiments using wild type and mutant forms of ArgR, combined with purified PepA, XerC and XerD showed that the ArgR149 mutant did not support recombination, but the ArgR5aa mutant did promote Holliday junction formation, raising the possibility that these two mutants interact differently with the Xer recombination machinery. Topology experiments showed that after adding topoisomerase 1, PepA is able to protect DNA from topoisomerase 1, and prevent ArgRWt binding to DNA. The two mutants ArgR149 and ArgR5aa are protecting DNA with more supercoiling. When PepA is added, migration profiles with the two mutants showed a binding problem with PepA. Other experiments involving the LEL triplet (leucine-glutamic acid-leucine) and amino-acids around it should be done in order to know the existence of a possible binding site for PepA.

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.