RESUMEN
Hmi1 is a UvrD-like DNA helicase required for the maintenance of the yeast Saccharomyces cerevisiae mitochondrial DNA (mtDNA). Deletion of the HMI1 ORF leads to the formation of respiration-deficient petite mutants, which either contain a short fragment of mtDNA arranged in tandem repeats or lack mtDNA completely. Here we characterize point mutants of the helicase designed to target the ATPase or ssDNA binding activity and show that these mutations do not separately lead to complete loss of the Hmi1 function. The mutant strains support ATP production via oxidative phosphorylation and enable us to directly analyze the impact of both activities on the stability of wild-type mtDNA in this petite-positive yeast. Our data reveal that Hmi1 mutants affecting ssDNA binding display a stronger defect in the maintenance of mtDNA compared to the mutants of ATP binding/hydrolysis. Hmi1 mutants impaired in ssDNA binding demonstrate sensitivity to UV irradiation and lower levels of Cox2 encoded by the mitochondrial genome. This suggests a complex and multifarious role for Hmi1 in mtDNA maintenance-linked transactions, some of which do not require the ATP-dependent helicase activity.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Adenosina Trifosfato/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Candida albicans , ADN Mitocondrial , Proteínas de Unión al ADN , Proteínas Fúngicas , Humanos , Candida albicans/genética , Candida albicans/metabolismo , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Proteínas Fúngicas/metabolismoRESUMEN
Irc3 is one of the six mitochondrial helicases described in Saccharomyces cerevisiae. Physiological functions of Irc3 are not completely understood as both DNA metabolic processes and mRNA translation have been suggested to be direct targets of the helicase. In vitro analysis of Irc3 has been hampered by the modest thermostability of the S. cerevisiae protein. Here, we purified a homologous helicase (Irc3op) of the thermotolerant yeast Ogataea polymorpha that retains structural integrity and catalytic activity at temperatures above 40 °C. Irc3op can complement the respiratory deficiency phenotype of a S. cerevisiae irc3Δ mutant, indicating conservation of biochemical functions. The ATPase activity of Irc3op is best stimulated by branched and double- stranded DNA cofactors. Single-stranded DNA binds Irc3op tightly but is a weak activator of the ATPase activity. We could also detect a lower level stimulation with RNA, especially with molecules possessing a compact three-dimensional structure. These results support the idea that that Irc3 might have dual specificity and remodel both DNA and RNA molecules in vivo. Furthermore, our analysis of kinetic parameters predicts that Irc3 could have a regulatory function via sensing changes of the mitochondrial ATP pool or respond to the accumulation of single-stranded DNA.
Asunto(s)
ADN Helicasas , Proteínas Fúngicas , Saccharomycetales , ADN/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , ARN , Saccharomyces cerevisiae , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Saccharomycetales/enzimología , Saccharomycetales/genéticaRESUMEN
Irc3 is a superfamily II DNA helicase required for the maintenance of mitochondrial DNA stability in Saccharomyces cerevisiae. Here, we show that recombinant Irc3 is a monomeric protein and that it can form a binary complex with forked DNA. The catalytically active enzyme is a monomer as no positive cooperativity of ATP hydrolysis or DNA unwinding can be detected. Interestingly, we find that Irc3 prefers to unwind the nascent lagging strand at a replication fork. Using DNase I footprinting, we demonstrate that Irc3 captures DNA substrates by establishing a strong contact at the DNA branching point. Additional protections on the lagging strand template suggest a 3'-to-5' polarity for Irc3 movement.
Asunto(s)
ADN Helicasas/metabolismo , Mitocondrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfatasas/metabolismo , Secuencia de Bases , ADN Helicasas/genética , ADN de Hongos/metabolismo , ADN Mitocondrial/metabolismo , Desoxirribonucleasa I/metabolismo , Hidrólisis , Modelos Biológicos , Peso Molecular , Proteínas Mutantes/metabolismo , Fenotipo , Mutación Puntual/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Irc3 is a superfamily II helicase required for mitochondrial DNA stability in Saccharomyces cerevisiae. Irc3 remodels branched DNA structures, including substrates without extensive single-stranded regions. Therefore, it is unlikely that Irc3 uses the conventional single-stranded DNA translocase mechanism utilized by most helicases. Here, we demonstrate that Irc3 disrupts partially triple-stranded DNA structures in an ATP-dependent manner. Our kinetic experiments indicate that the rate of ATP hydrolysis by Irc3 is dependent on the length of the double-stranded DNA cosubstrate. Furthermore, the previously uncharacterized C-terminal region of Irc3 is essential for these two characteristic features and forms a high affinity complex with branched DNA. Together, our experiments demonstrate that Irc3 has double-stranded DNA translocase activity.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Hongos/metabolismo , ADN Mitocondrial/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , ADN Helicasas/genética , ADN de Hongos/química , ADN de Hongos/genética , ADN Mitocondrial/química , ADN Mitocondrial/genética , Hidrólisis , Cinética , Mitocondrias/metabolismo , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.
Asunto(s)
ADN Polimerasa gamma/fisiología , Desoxirribonucleótidos/fisiología , Genoma Mitocondrial/fisiología , Mitocondrias/fisiología , Saccharomyces cerevisiae/fisiología , Núcleo Celular/fisiología , Citoplasma/fisiología , Reparación de la Incompatibilidad de ADN/fisiología , Replicación del ADN/fisiología , ADN Mitocondrial/metabolismo , Inestabilidad GenómicaRESUMEN
Integrity of mitochondrial DNA (mtDNA) is essential for cellular energy metabolism. In the budding yeast Saccharomyces cerevisiae, a large number of nuclear genes influence the stability of mitochondrial genome; however, most corresponding gene products act indirectly and the actual molecular mechanisms of mtDNA inheritance remain poorly characterized. Recently, we found that a Superfamily II helicase Irc3 is required for the maintenance of mitochondrial genome integrity. Here we show that Irc3 is a mitochondrial DNA branch migration enzyme. Irc3 modulates mtDNA metabolic intermediates by preferential binding and unwinding Holliday junctions and replication fork structures. Furthermore, we demonstrate that the loss of Irc3 can be complemented with mitochondrially targeted RecG of Escherichia coli. We suggest that Irc3 could support the stability of mtDNA by stimulating fork regression and branch migration or by inhibiting the formation of irregular branched molecules.
Asunto(s)
ADN Helicasas/metabolismo , ADN Mitocondrial/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , ADN Cruciforme/química , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , ADN Mitocondrial/química , ADN Mitocondrial/genética , Glucosa/metabolismo , Mitocondrias/química , Mitocondrias/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability.
Asunto(s)
Adenosina Trifosfatasas/fisiología , ADN Helicasas/fisiología , ADN Mitocondrial/metabolismo , Genoma Mitocondrial , Mitocondrias/enzimología , Proteínas Mitocondriales/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Mitocondrial/química , ARN Polimerasas Dirigidas por ADN/genética , Epistasis Genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.
Asunto(s)
Candida/metabolismo , Replicación del ADN/fisiología , ADN de Hongos/metabolismo , ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Recombinación Genética/fisiología , Candida/genética , ADN de Hongos/genética , ADN Mitocondrial/genética , Mitocondrias/genética , Telómero/genética , Telómero/metabolismoRESUMEN
Mitochondrial metabolism is targeted by conserved signaling pathways that mediate external information to the cell. However, less is known about whether mitochondrial dysfunction interferes with signaling and thereby modulates the cellular response to environmental changes. In this study, we analyzed defective filamentous and invasive growth of the yeast Saccharomyces cerevisiae strains that have a dysfunctional mitochondrial genome (rho mutants). We found that the morphogenetic defect of rho mutants was caused by specific downregulation of FLO11, the adhesin essential for invasive and filamentous growth, and did not result from general metabolic changes brought about by interorganellar retrograde signaling. Transcription of FLO11 is known to be regulated by several signaling pathways, including the filamentous-growth-specific MAPK and cAMP-activated protein kinase A (cAMP-PKA) pathways. Our analysis showed that the filamentous-growth-specific MAPK pathway retained functionality in respiratory-deficient yeast cells. In contrast, the cAMP-PKA pathway was downregulated, explaining also various phenotypic traits observed in rho mutants. Thus, our results indicate that dysfunctional mitochondria modulate the output of the conserved cAMP-PKA signaling pathway.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , ADN Polimerasa I/genética , ARN Polimerasas Dirigidas por ADN/genética , Regulación hacia Abajo , Sistema de Señalización de MAP Quinasas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Mutación , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
We developed m:Explorer for identifying process-specific transcription factors (TFs) from multiple genome-wide sources, including transcriptome, DNA-binding and chromatin data. m:Explorer robustly outperforms similar techniques in finding cell cycle TFs in Saccharomyces cerevisiae. We predicted and experimentally tested regulators of quiescence (G0), a model of ageing, over a six-week time-course. We validated nine of top-12 predictions as novel G0 TFs, including Δmga2, Δcst6, Δbas1 with higher viability and G0-essential TFs Tup1, Swi3. Pathway analysis associates longevity to reduced growth, reprogrammed metabolism and cell wall remodeling. m:Explorer (http://biit.cs.ut.ee/mexplorer/) is instrumental in interrogating eukaryotic regulatory systems using heterogeneous data.
Asunto(s)
Algoritmos , Modelos Logísticos , Viabilidad Microbiana/genética , Saccharomyces cerevisiae/genética , Sitios de Unión , Ciclo Celular , Pared Celular/genética , Pared Celular/metabolismo , Biología Computacional/métodos , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Internet , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.
Asunto(s)
ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , Replicación del ADN , ADN de Hongos/metabolismo , ADN Mitocondrial/metabolismo , Mitocondrias/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Biocatálisis , Exonucleasas/metabolismo , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Unión Proteica , Alineación de Secuencia , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
Polymerase γ is solely responsible for fast and faithful replication of the mitochondrial genome. High processivity of the polymerase γ is often achieved by association of the catalytic subunit with accessory factors that enhance its catalytic activity and/or DNA binding. Here we characterize the intrinsic catalytic activity and processivity of the recombinant catalytic subunit of yeast polymerase γ, the Mip1 protein. We demonstrate that Mip1 can efficiently synthesize DNA stretches of up to several thousand nucleotides without dissociation from the template. Furthermore, we show that Mip1 can perform DNA synthesis on double-stranded templates utilizing a strand displacement mechanism. Our observations confirm that in contrast to its homologues in other organisms, Mip1 can function as a single-subunit replicative polymerase.
Asunto(s)
ADN Polimerasa I/metabolismo , ADN Mitocondrial/metabolismo , Mitocondrias/enzimología , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Dominio Catalítico , ADN Polimerasa I/química , ADN Polimerasa I/genética , ADN Polimerasa I/aislamiento & purificación , Replicación del ADN , ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
DNA replication origins are licensed in early G(1) phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G(1) to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G(1) might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase.
Asunto(s)
Replicación del ADN/fisiología , ADN de Hongos/biosíntesis , Origen de Réplica/fisiología , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/fisiología , ADN Helicasas , ADN de Hongos/genética , Fase G1/fisiología , Fase S/fisiología , Saccharomyces cerevisiae/genéticaRESUMEN
Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast.
Asunto(s)
Candida albicans/genética , Replicación del ADN/fisiología , ADN Mitocondrial/biosíntesis , Secuencias Invertidas Repetidas/genética , Recombinación Genética/fisiología , Enzimas de Restricción del ADN/metabolismo , ADN Concatenado/genética , ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Agar , Electroforesis en Gel Bidimensional , Dosificación de Gen/genética , Estructura Molecular , ARN/genética , ARN/metabolismo , Origen de Réplica/fisiología , Mapeo RestrictivoRESUMEN
Mitochondria of eukaryotic organisms contain populations of DNA molecules that are packed into higher-order structures called mitochondrial nucleoids (mt-nucleoids). In Saccharomyces cerevisiae, the compaction of mitochondrial DNA (mtDNA) into mt-nucleoids is mediated primarily by the high-mobility group (HMG) box-containing protein Abf2, which is an important player in stabilization and metabolism of mtDNA. Although it is evident that analogous proteins must exist in other yeast species, an apparently fast divergence rate has precluded their identification, characterization and comparative analysis. Using in silico analysis of the complete genome sequence of the pathogenic yeast Candida albicans we predicted that the ORF 19.400/19.8030 assigned as GCF1 encodes a putative mitochondrial HMG box-containing protein. In contrast to Abf2p, which contains two HMG boxes, Gcf1p contains only one C-terminal HMG box. In addition, it contains one putative coiled-coil domain with a potential role in protein dimerization. Fluorescence microscopy analysis of a C-terminally tagged Gcf1p with green fluorescent protein (GFP) revealed its mitochondrial localization in both heterologous (S. cerevisiae) and native (C. albicans) hosts. Biochemical analyses of DNA-binding properties indicate that Gcf1p is, similarly to Abf2p, a non-specific DNA-binding protein. To analyse the role of Gcf1p in mtDNA metabolism, we constructed strains lacking one functional allele of the GCF1 gene and carrying one GCF1 allele under the control of the MET3 promoter. Under repressible conditions this strain exhibited a more than 3000-fold decrease in levels of GCF1 mRNA, which was correlated with a substantial decrease in the number of mtDNA copies as well as recombination intermediates. The dramatic effect of reduced levels of Gcf1p on mtDNA metabolism indicates that the protein is involved in essential molecular transactions that relate to the mitochondrial genome.
Asunto(s)
Candida albicans/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Candida albicans/genética , Candida albicans/fisiología , ADN de Hongos/metabolismo , ADN Mitocondrial/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HMGB/química , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Microscopía Fluorescente , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia MolecularRESUMEN
The mechanistic details of mtDNA maintenance in petite-negative yeasts have remained largely unexplored. We report here that the DNA helicase Hmi1p plays a crucial role in mtDNA stability in Candida albicans. Like its counterpart in Saccharomyces cerevisiae, Hmi1p in C. albicans (CaHmi1p) contains a C-terminal mitochondrial targeting signal that is functional in both organisms. Biochemical analysis demonstrates that CaHmi1p is a protein possessing ATP-dependent 3'-5' DNA-unwinding activity. Deletion of both HMI1 alleles does not lead to complete loss of mtDNA in C. albicans; however, substantial fragmentation of the wild-type mitochondrial genome, reduction of mtDNA mass and loss of wild-type nucleoid distribution occur. Specific regions of the mitochondrial genome give rise to mtDNA molecule populations with altered characteristics upon CaHMI1 deletion. Fragmentation of the mitochondrial genome can be reversed by reintroduction of CaHmi1p. This is the first time that a gene required for wild-type mtDNA maintenance in S. cerevisiae has been demonstrated to be nonessential in a petite-negative yeast.
Asunto(s)
Candida albicans/enzimología , ADN Helicasas/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Regulación Fúngica de la Expresión Génica , Adenosina Trifosfato/metabolismo , Transporte Biológico , ADN Helicasas/química , ADN Helicasas/genética , ADN Mitocondrial/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microscopía Fluorescente , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Proteínas Mitocondriales , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.
Asunto(s)
ADN Helicasas/fisiología , Mitocondrias/enzimología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Secuencia de Bases , ADN/química , ADN/metabolismo , ADN Mitocondrial/metabolismo , ADN de Cadena Simple/química , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Glicerol/química , Hidrólisis , Cinética , Proteínas Mitocondriales , Modelos Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Nucleótidos/química , Oligonucleótidos/química , Plásmidos/metabolismo , Unión Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Recombinación Genética , Especificidad por Sustrato , Factores de TiempoRESUMEN
Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates rho(-) and rho(0) cells. Here we demonstrate that, in rho(-) yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric rho(-) mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1-5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1Delta yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.