5,411 research outputs found
Structural and functional analysis of proteins that regulate fission yeast telomerase activity
Shelterin is a protein-DNA complex found at chromosome ends, which prevents their recognition by the double strand break surveillance function of the DNA damage response and plays important roles in regulation of telomere assembly and telomerase activity. In Schizosaccharomyces pombe, the shelterin complex consists of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1. In spite of its biological importance in maintaining genomic integrity, relatively little is known about the structures of the proteins of the shelterin complex and how their interactions coordinate their regulatory functions.
This thesis describes experiments to investigate the structural and biochemical basis of shelterin assembly and regulation. Using a combination of approaches, Ccq1 was shown to behave as an elongated, modular and somewhat flexible molecule, dimerising tightly through a C-terminal coiled-coil region. Binding experiments showed that Ccq1 interacts with Tpz1 with micromolar affinity, whereas Poz1 and Tpz1 were found to bind more tightly with an apparent Kd in the low nanomolar range. This suggests a highly stable Poz1-Tpz1 interaction occurs in vivo in keeping with the proposed role for this sub-complex as a structural bridge within the shelterin assembly. The minimal binding motifs for Poz1 and Ccq1 were identified within the C-terminal Tpz1 region and, importantly, shown to not overlap, allowing each protein to bind to Tpz1 non- competitively to form a stable three component sub-complex. Negative stain
electron microscopy of this tripartite assembly containing full-length Poz1-Tpz1-Ccq1, co-expressed in insect cells, generated a low resolution envelope in which the three components are arranged to form an interrupted shell-like structure surrounding a central void. Most strikingly, the structure shows a markedly asymmetric architecture in spite of the 2: 2: 2 stoichiometry of Ccq1, Tpz1 and Poz1 in the complex. The molecular basis for this asymmetry remains unclear, but may have important implications for telomerase regulation.Open Acces
The Development of a Dual-Tag Affinity Purification System and its Application to Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1
Protein-protein interactions (PPI) play a vital role in almost every cellular process. Although many methodologies exist to probe PPIs, one of the most successful and widely employed is tandem affinity purification coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). Although best demonstrated in yeast, TAP has encountered significant hurdles in its application to mammalian systems, especially the observed low yield of bait protein and its interacting partners after two consecutive purifications.
To address these issues, a novel dual-tag affinity purification (DAP) system was developed that not only enhances bait protein recovery, but also allows for rapid evaluation of dual-tag compatibility with a protein of interest based on its known subcellular localization. In addition, several tags of varying composition were constructed to allow for maximal bait protein compatibility. With this system, mammalian bait protein yield was improved by more than 200% relative to previously published results.
Capitalizing on this success, DAP was applied to human telomere binding proteins TRF1, TRF2, and POT1 to garner a greater understanding of the protein networks that involve the telomere. Expectedly, all the members of the telosome complex were identified at frequencies that lend evidence towards the currently accepted architecture. Also identified were several other novel proteins and subcomplexes that may enhance our understanding of telomere maintenance / length regulation. For instance, members of the classical nuclear import system co-purified with both TRF1 and TRF2. Although previously documented for TRF1, TRF2’s association with importin alpha (KPNA2) and beta (KPNB1) has not been demonstrated till now. Interestingly, further study revealed that KPNA2 acts as a negative regulator of TRF2 nuclear localization.
This observation could have far-reaching implications as TRF2 is thought to be also heavily involved in the DNA damage response. Along these lines, a more indepth MS analysis revealed several putative phosphorylation sites along TRF2’s sequence. One site, pS380, seems to be phosphorylated by the DNA-damage kinase ATM and plays a role in a cell’s proliferative capacity, possibly affecting telomere length regulation. The studies contained here within demonstrate the efficacy of DAP-LC-MS/MS to provide useful leads with regards to the study of PPIs
Purification of G4 Resolvase1 : a protein entangled in many cellular functions
G-quadruplexes are supersecondary structures that form in guanine-rich regions of DNA and
RNA. These structures are extremely thermally stable, likely to form in ~750,000 locations in the
human genome and are enriched in the promoter regions of proto-oncogenes and developmental
genes, non-coding regions, and within telomeres. Due to the prevalence of these structures in key
genomic locations, these structures are cable of regulating a large fraction of cellular processes.
Within humans, the enzyme G4 Resolvase 1 (G4R1) is responsible for the majority of Gquadruplex
resolving activity. This places G4R1 at the forefront of regulating all functions
involved with G-quadruplexes. Despite regulating G-quadruplexes, a nearly ubiquitous structure
throughout the human genome, G4R1 is relatively understudied. Within this thesis, I present a
method for producing highly pure and selectively catalytically active samples of rG4R1 and
discuss the potential involvement of G4R1 with the pathology of Amyotrophic Lateral Sclerosis.Thesis (B.?)Honors Colleg
テロメア及びテロメラーゼを標的としたがん治療法及びがん診断法の開発
テロメラーゼによるテロメアDNA伸長反応(テロメラーゼ反応)は細胞のがん化を導く。そこで本研究では、テロメラーゼ反応の阻害によるがん治療法及び、 テロメラーゼ活性測定を介したがん診断法の開発を行った。1.がん治療法の開発:テロメアDNAにより形成されるG-quadruplex構造に結合するリガンドは、テロメラーゼ反応を阻害することが知られている。しかし従来のG-quadruplexリガンドの多くは、細胞内では染色体二本鎖DNAとも非特異的に結合するため、競合的にG-quadruplexへの結合が阻害される。さらに最近、細胞内の分子クラウディング(Molecular Crowding:MC)環境による水の活量低下も、G-quadruplexリガンドの結合を阻害することが報告された。そこでこれら阻害要因に対するG-quadruplexリガンドの官能基、中心金属、π平面の大きさの影響について系統的に解析した。その結果、アニオン性官能基を有するフタロシアニンは、 二本鎖DNAとは静電的に反発するため、G-quadruplexへの特異性が高いことが見出された。さらにアニオン性フタロシアニンはG-quadruplexに結合する際に水分子を取り込まないため、水の活量低下によるG-quadruplexへの結合阻害が生じないことが示された。そのためアニオン性フタロシアニンは、大量の二本鎖DNA存在下 及びMC条件下においてもテロメラーゼ反応を効率よく阻害することが示された。以上の結果は、アニオン性フタロシアニンが細胞核内においても効率よくテロメアDNAのG-quadruplexに結合し、テロメラーゼ反応を阻害することを示唆している。2.がん診断法の開発:従来のテロメラーゼ活性測定技術は、テロメラーゼ反応とPolymerase Chain Reaction(PCR)を組み合わせた手法である。そのため生体試料中のPCR阻害分子によって擬陰性結果が生じることがあった。そこで本研究では、テロメラーゼ反応後の反応産物を磁性ビーズ上に固定化、洗浄することで、PCR阻害分子を取り除く手法を開発した。さらにその後に行うPCRの産物をサイクリングプローブ法によって高感度に検出する方法を開発した。その結果、これらの手法を組み合わせることによってPCR阻害分子である胆汁酸、ビリルビン、ヘモグロビン存在下においても、阻害の影響を受けることなくがん細胞中のテロメラーゼ活性を測定できることが示された。また、検出感度50細胞という非常に高い感度でがん細胞を検出できることが示された。甲南大学平成25年度(2013年度
Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors
The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3′-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties
Contribution of non-canonical DNA G-quadruplex structures to premature ageing
Previous studies have identified Cockayne Syndrome B (CSB) as a helicase that can resolve non-canonical DNA structures, called G-quadruplexes (G4s). The aim of this study is to investigate the properties of CSB as a G4-binder and -resolvase, and examine the correlation between the G4-helicase activity of CSB and premature ageing phenotype observed in CSB-deficient cells. Accordingly, the recombinant CSB full-length protein (FL) and its helicase- “like" domain (HD) were respectively expressed from insect and bacterial cells, and their resolvase and binding activities were tested over a large panel of DNA substrates. Native gel analysis and biophysical characterisations revealed that ribosomal DNA (rDNA) sequences, that typically act as CSB substrate, can form intermolecular G4s. We discovered that intermolecular G4s were strongly bound by CSB with picomolar affinity, whilst negligible binding to intramolecular G4s was observed. In vitro and cellular data demonstrated that G4-ligands can compete with CSB for binding to intermolecular rDNA G4, which results in CSB being displaced off the nucleoli of cells upon treatment with G4-ligands. Immunostaining with the selective G4-antibody BG4 revealed a lack of BG4-staining in the nucleoli of CSB-deficient cells after exogenous expression of recombinant CSB, further corroborating the hypothesis that CSB can bind intermolecular rDNA G4s in the nucleoli and compete with BG4 for the binding of such DNA-substrate. The work presented in this thesis allowed us to observe that (I) intermolecular G4s are likely to form from long-range distant rDNA sequences within the nucleoli of cells, and (II) CSB specifically binds and resolves these structures. Our results provide the first evidence of an endogenous protein that specifically interacts with intermolecular G4s, suggesting potential biological significance of these structures. The biological relevance of intermolecular rDNA G4s could be key in rare genetic disorders like Cockayne Syndrome, where senescence and premature ageing is observed when CSB is functionally mutated.Open Acces
Biosynthesis of a G-Quadruplex—forming sequence and its stabilization by ligands
In addition to the Watson and Crick B-form duplex DNA, G-quadruplexes are four-stranded DNA structures formed in vivo by the self-assembly of guanine-rich sequences. These can be formed by one, two or four separate strands of DNA and present a diversity of topologies, defined by the strand orientation, loop size and sequence. G-quadruplexes can be found in telomeres, immunoglobulin switch regions and gene promoter regions. The biological relevant location on the genome makes these high-order structures an attractive target for drug design and the development of highly specific ligands that bind and stabilize G-quadruplex with therapeutic activity. Herein, the biosynthesis of a novel G-rich quadruplex-forming DNA sequence 58Sγ3 is described by plasmid amplification. The recovery and purification of 58Sγ3 oligonucleotide using size-exclusion chromatography is presented. The G-quadruplex formation is promoted and its topology is determined by circular dichroism. The stabilization of the G-quadruplex structure with quinoline and naphthalene-based derivatives is studied using melting analysis, G4-FID and PCR-stop assays. The results suggest that 58Sγ3 folds into a parallel-stranded G-quadruplex structure in 500 mM KCl buffer and that naphthalene-based ligands bind and stabilize the G-quadruplex structure. The ligands are also found to be quadruplex-specific over duplex DNA and inhibit Taq DNA polymerase. This work provides evidence for G-quadruplex formation within the immunoglobulin switch regions. Furthermore, it is suggested that the novel ligands here reported act as potent specific G-quadruplex binders and may also potentially be used to inhibit genes transcription in tumor cells.Além da forma B Watson e Crick do ADN duplex, os G-quadruplexes são estruturas de ADN de quatro cadeias, formadas in vivo pela auto-associação de sequências ricas em guaninas. Estas podem ser formadas por uma, duas ou quatro cadeias distintas de ADN e apresentar uma diversidade de topologias, definidas pela orientação da cadeia, tamanho dos loops e a sequência. G-quadruplexes podem ser encontrados nos telómeros, regiões de troca das imunoglobulinas e nas regiões dos promotores génicos. A localização biologicamente relevante no genoma faz com que estas estruturas altamente ordenadas sejam um alvo atrativo do desenho de fármacos e o desenvolvimento de ligandos altamente específicos que ligam e estabilizam o G-quadruplex com ação terapêutica. Neste trabalho, descreve-se a biossíntese da nova sequência de ADN rica em guaninas e formadora de G-quadruplex 58Sγ3, utilizando amplificação por plasmídeo. A recuperação e purificação do oligonucleótido 58Sγ3 é efetuada por cromatografia de exclusão molecular. A formação de G-quadruplex é promovida e a sua topologia é determinada por dicroísmo circular. A estabilização da estrutura do G-quadruplex com ligandos derivados de quinolina e naftaleno é estudada utilizando ensaios de estabilização térmica no dicroísmo circular, G4-FID e PCR-stop. Os resultados sugerem que 58Sγ3 adota uma estrutura G-quadruplex paralela em tampão 500 mM KCl e que os ligandos de naftaleno ligam e estabilizam a estrutura do G-quadruplex. Os ligandos demonstraram também ser específicos do G-quadruplex em relação ao ADN duplex além de inibir a Taq ADN polimerase. Este trabalho fornece evidência da formação de G-quadruplex nas regiões de troca das imunoglobulinas. Além disso, sugere que os derivados de naftaleno atuam como ligandos do G-quadruplex e que podem ser potencialmente utilizados para inibir a transcrição de genes em células tumorais
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