The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a highmobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4- dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria

Structural characterization of a mitochondrial dna maintenance protein of biomedical interest

En aquest treball de tesi presentem la caracterització estructural de la proteïna de 'Candida albicans' ('C.albicans') Gcf1p. Gcf1p és una proteïna d'unió a l'ADN, que localitza al mitocondri de 'C.albicans' i que és essencial per al manteniment de l'ADN mitocondrial (ADNmt) així com per a la supervivència del microorganisme. 'C.albicans' és un fong dimòrfic amb capacitat de crèixer en hifes invasives i causar patologia en humans. 'C.albicans' forma part de la flora microbiana en individus sans, no obstant, pot esdevenir patògen oportunista causant de infeccions superficials (candidiasi) així com d'infeccions invasives (candidèmia). El creixent nombre de casos de candidèmia, derivats principalment d'infeccions d'origen nosocomial en hospitals, així com la seva elevada mortalitat associada , al voltant del 50%, fa de 'C.albicans' una microorganisme d'interès biomèdic. Per altra banda, l'adquisició de resistència als tractaments antifúngics convencionals per part de 'C.albicans' i d'espècies properes ('Candida auris') fa urgent la necessitat d'una descripció dels mecanismes replicatius i invasius d'aquestes espècies. En aquesta línia, una millor descripció dels mecanismes fonamentals en el manteniment de l'ADNmt és un potencial punt de partida per a la recerca de nous fàrmacs específics. Aquest treball de tesi aporta informació novedosa sobre com la proteïna de 'C.albicans' Gcf1p uneix i compacta l'ADNmt. Per altra banda, aquest treball de tesi aporta informació novedosa en el camp de la biologia evolutiva. L'origen i evolució dels mitocondris des de organismes independents a orgànuls cel·lulars (teoria endosimbiòtica) és acceptada per la comunitat científica com a punt clau en l'aparició d'organismes eucariotes (protists, fongs, plantes i animals). Els mecanismes de compactació i replicació de l'ADNmt presenta alta variabilitat inter i intra-regnes. La proteïna Gcf1p està relacionada amb la replicació depenent de recombinació que està present en l'ADNmt de 'C.albicans' i que és completament diferent al model de replicació de l'ADNmt en mamífers. La informació aportada per aquest treball respecte a la unió i compactació de l'ADN per part de Gcf1p obre un punt de partida per a comparar la variabilitat en la organització de l'ADNmt de C.albicans amb la d'altres llevats (principalment 'Saccharomyces cerevisiae' ('S.cerevisiae') així com les diferències amb organismes llunyans en l'evolució ('Homo sapiens' ('H.sapiens')). Aquest treball és també un potencial punt de partida per a entendre millor la divergència evolutiva observada en eucariotes. En aquest treball s'han usat tècniques de biologia molecular per al clonatge en vectors plasmídics i l'expressió heteròloga de proteïnes de llevat en 'Escherichia coli'. Així mateix, s'han usat tècniques de purificació de proteïnes i s'ha aconseguit optimitzar un protocol de producció compatible en termes de rendiment i puresa amb l'anàlisi per mètodes de biologia estructural, principalment cristal·lografia de macromolècules (MX), dispersió de rajos X a angle petit (SAXS) i microscopia electrònica (EM). Els resultats obtinguts per aquestes tres tècniques aporten observacions complementaries sobre la interacció de Gcf1p amb l'ADN. En suma, aquest treball de tesi aporta evidències sòlides que indiquen que el mecanisme de reconeixement de l'ADNmt en 'C.albicans' presenta diferències fonamentals amb els descrits per 'S.cerevisiae' i 'H.sapiens'. Els nostres resultats suposen un important avenç en la descripció d'un procès essencial per als organismes eucariotes com és la organització i manteniment de l'ADN mitocondrial.En este trabajo de tesis se presenta la caracterización estructural de la proteína de 'Candida albicans' ('C.albicans') Gcf1p. Gcf1p es una proteína de unión al ADN, que transloca a la mitocondria de 'C.albicans' y que es esencial para el mantenimiento del ADN mitocondrial (ADNmt) así como para la supervivencia del organismo. 'C.albicans' es un hongo dimórfico con capacidad de formar hifas invasivas y causar patología en humanos. 'C.albicans' forma parte de la flora microbiana en individuos sanos, no obstante, puede devenir patógeno oportunista causant de infecciones superficiales (candidiasis) así como de infecciones invasivas (candidemia). El crecimiento del número de casos de candidemia, derivados principalmente de infecciones nosocomiales, así como la elevada tasa de mortalidad asociada (alrededor del 50 %), hace de 'C.albicans' un organismo de elevado interés biomédico. Por otro lado, la adquisición de resistencia a los tratamientos antifúngicos más comunes por parte de 'C.albicans' y especies relacionadas ('Candida auris') hace urgente la necesidad de una descripción de los mecanismos replicativos e invasivos de estas especies. En la misma línia, una mejor descripción de los mecanismos fundamentales en el mantenimiento del ADNmt es un potencial punto de partida para la investigación de nuevos fármacos específicos. En este trabajo de tesis se aporta información novedosa sobre como la proteína de 'C.albicans' Gcf1p une y compacta el ADNmt. Por otro lado, este trabajo de tesis aporta información novedosa en el campo de la biología evolutiva. El origen y evolución de las mitocondrias desde organismos independientes a orgánulos celulares (teoría endosimbiótica) es aceptada por la comunidad científica como punto de partida en la aparición de organismos eucariotas (protistas, hongos, plantas y animales). Los mecanismos de compactación y replicación del ADNmt presentan alta variabilidad inter e intra-reinos. La proteína Gcf1p está relacionada con la replicación dependiente de recombinación presente en 'C.albicans' y que es completamente diferente al modelo de replicación del ADNmt en mamíferos. La información aportada en este trabajo de tesis acerca de la unión y compactación del ADN por parte de Gcf1p supone un punto de partida para comparar la variabilidad en la organización del ADNmt en 'C.albicans' respecto otras levaduras (principalmente 'Saccharomyces cerevisiae' ('S.cerevisiae') así como respecto de otros organismos evolutivamente distantes ('Homo sapiens' (H.sapiens')). Asimismo, este trabajo supone también un potencial punto de partida para entender la divergencia evolutiva observada entre eucariotas. En este trabajo se han usado técnicas de biologia molecular para el clonaje en vectores plasmídicos y la expresión heteróloga de proteínas de levadura en 'Escherichia coli'. Asimismo, se han usado técnicas de purificación de proteínas logrando optimizar un protocolo de producción compatible en términos de rendimiento y pureza con el análisis por métodos de biología estructural, principalmente cristalografía de macromoléculas (MX), dispersión de rayos X en ángulo pequeño (SAXS) y microscopia electrónica (EM). Los resultados obtenidos por estas tres técnicas aportan observaciones complementarias sobre la interacción de Gcf1p con el ADN. En su conjunto, este trabajo de tesis aporta evidencias que indican que el mecanismo de reconocimiento del ADNmt en 'C.albicans' presenta diferencias fundamentales respecto de los descritos para 'S.cerevisiae' y 'H.sapiens'. Nuestros resultados suponen un importante avance en la descripción de un proceso esencial para los organismos eucariotas como es la organización y mantenimiento del ADN mitocondrial.This thesis work is centred in the structural characterisation of Candida albicans (C.albicans) Gcf1p. Gcf1p is a DNA-binding protein located in C.albicans mitochondria that is essential for mitochondrial DNA (mtDNA) maintenance as well as for the viability of such organism. C.albicans is a dimorphic yeast with the capability to form invasive hyphal structures causing pathology in humans. C.albicans is a part of the mycobiota in healthy individuals. Nevertheless, it can be causing both superficial infections (candidiasis) as well as invasive infections (candidemia). Growing prevalence of candidemia, mainly caused by nosocomial transmission in hospital, together with the its high lethality (about 50% mortality rate) makes C.albicans a potential biomedical target of high interest. In addition, C.albicans and related species (Candida auris) display increasing resistance to the conventional antifungal treatments. Regarding this point, a better understanding of the fundamental mechanisms involved in mtDNA maintenance is a potential starting point for drug discovery. This thesis work provides novel information regarding how Gcf1p binds and compacts mtDNA. This thesis work also provides with novel information that can be of high interest in evolutionary biology. Origin and evolution of mitochondria from independent organisms to cell organelles (endosymbiotic theory) is broadly regarded by the scientific community as a key point in the apparition of eukaryotes (protists, fungi, plants and animals). The mechanisms behind mtDNA compaction and replication shows a high diversity both inter-reigns and intra-reigns. Gcf1p is also related with recombination-driven-replication mechanism present in C.albicans, which is completely different to the current model for mtDNA replication in mammals. The results provided by this thesis work in regard the union and compaction of DNA by Gcf1p suppose also a starting point for comparing the mtDNA in Candida albicans in regard of other yeast (mainly Saccharomyces cerevisiae (S.cerevisiae)) as well as in regard of distant organisms (Homo sapiens (H.sapiens)). It is also a starting point to understand evolutionary divergence amongst eukaryotes. This work has made use of molecular biology techniques for the cloning in plasmid vector, as well as, for the heterologous expression of yeast proteins in Escherichia coli. In addition, protein purification techniques have been applied obtaining an optimized production protocol compatible with the experimental analysis by means of structural biology methods, mainly macromolecular crystallography (MX), Small-Angle X-ray Scattering (SAXS) and Electron Microscopy (EM). Results from these three techniques provide complementary evidences about the interaction of Gcf1p with DNA. In summary, this tesis work provides evidences that indicate that the mtDNA recognition mechanism in C.albicans presents fundamental differences regarding those described for S.cerevisiae and H.sapiens. Our results suppose an important advance in the description of spatial organization of mitochondrial DNA, an essential process eukaryotic organism.Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicin

The mutation R107Q alters mtSSB ssDNA compaction ability and binding dynamics

International audienceAbstract Mitochondrial single-stranded DNA-binding protein (mtSSB) is essential for mitochondrial DNA (mtDNA) replication. Recently, several mtSSB variants have been associated with autosomal dominant mitochondrial optic atrophy and retinal dystrophy. Here, we have studied at the molecular level the functional consequences of one of the most severe mtSSB variants, R107Q. We first studied the oligomeric state of this variant and observed that the mtSSBR107Q mutant forms stable tetramers in vitro. On the other hand, we showed, using complementary single-molecule approaches, that mtSSBR107Q displays a lower intramolecular ssDNA compaction ability and a higher ssDNA dissociation rate than the WT protein. Real-time competition experiments for ssDNA-binding showed a marked advantage of mtSSBWT over mtSSBR107Q. Combined, these results show that the R107Q mutation significantly impaired the ssDNA-binding and compacting ability of mtSSB, likely by weakening mtSSB ssDNA wrapping efficiency. These features are in line with our molecular modeling of ssDNA on mtSSB showing that the R107Q mutation may destabilize local interactions and results in an electronegative spot that interrupts an ssDNA-interacting-electropositive patch, thus reducing the potential mtSSB-ssDNA interaction sites.</jats:p

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compactio

Structural analysis of the Candida albicans mitochondrial DNA maintenance factor Gcf1p reveals a dynamic DNA-bridging mechanism

International audienceAbstract The compaction of mitochondrial DNA (mtDNA) is regulated by architectural HMG-box proteins whose limited cross-species similarity suggests diverse underlying mechanisms. Viability of Candida albicans, a human antibiotic-resistant mucosal pathogen, is compromised by altering mtDNA regulators. Among them, there is the mtDNA maintenance factor Gcf1p, which differs in sequence and structure from its human and Saccharomyces cerevisiae counterparts, TFAM and Abf2p. Our crystallographic, biophysical, biochemical and computational analysis showed that Gcf1p forms dynamic protein/DNA multimers by a combined action of an N-terminal unstructured tail and a long helix. Furthermore, an HMG-box domain canonically binds the minor groove and dramatically bends the DNA while, unprecedentedly, a second HMG-box binds the major groove without imposing distortions. This architectural protein thus uses its multiple domains to bridge co-aligned DNA segments without altering the DNA topology, revealing a new mechanism of mtDNA condensation

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247–258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247–258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compactio

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compactio