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1.
Nucleic Acids Res ; 52(10): 5912-5927, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38742632

RESUMO

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.


Assuntos
DNA de Cadeia Simples , Proteínas de Ligação a DNA , Mutação , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/química , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína
2.
Methods Mol Biol ; 2615: 121-137, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36807789

RESUMO

Mitochondrial transcription factor A (TFAM) plays a key role in the organization and compaction of the mitochondrial genome. However, there are only a few simple and accessible methods available to observe and quantify TFAM-dependent DNA compaction. Acoustic Force Spectroscopy (AFS) is a straightforward single-molecule force spectroscopy technique. It allows one to track many individual protein-DNA complexes in parallel and to quantify their mechanical properties. Total internal reflection fluorescence (TIRF) microscopy is a high-throughput single-molecule technique that permits the real-time visualization of the dynamics of TFAM on DNA, parameters inaccessible with classical biochemistry tools. Here we describe, in detail, how to set up, perform, and analyze AFS and TIRF measurements to study DNA compaction by TFAM.


Assuntos
DNA , Fenômenos Mecânicos , DNA/química , Proteínas Mitocondriais/genética , Microscopia de Fluorescência/métodos , Análise Espectral/métodos , Acústica , DNA Mitocondrial/genética
3.
Biochim Biophys Acta Mol Basis Dis ; 1868(10): 166467, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35716868

RESUMO

Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression, and packaging of mitochondrial DNA (mtDNA). Recently, a pathogenic homozygous variant in TFAM (P178L) has been associated with a severe mtDNA depletion syndrome leading to neonatal liver failure and early death. We have performed a biochemical characterization of the TFAM variant P178L in order to understand the molecular basis for the pathogenicity of this mutation. We observe no effects on DNA binding, and compaction of DNA is only mildly affected by the P178L amino acid change. Instead, the mutation severely impairs mtDNA transcription initiation at the mitochondrial heavy and light strand promoters. Molecular modeling suggests that the P178L mutation affects promoter sequence recognition and the interaction between TFAM and the tether helix of POLRMT, thus explaining transcription initiation deficiency.


Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
New Phytol ; 225(1): 340-355, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31469444

RESUMO

Awns, bristle-like structures extending from grass lemmas, provide protection against predators, contribute to photosynthesis and aid in grain dispersal. In wheat, selection of awns with minimal extension, termed awnletted, has occurred during domestication by way of loci that dominantly inhibit awn development, such as Tipped1 (B1), Tipped2 (B2), and Hooded (Hd). Here we identify and characterize the B1 gene. B1 was identified using bulked segregant RNA-sequencing of an F2 durum wheat population and through deletion mapping of awned bread wheat mutants. Functional characterization was accomplished by gene overexpression while haplotype analyses assessed B1 polymorphisms and genetic variation. Located on chromosome 5A, B1 is a C2H2 zinc finger encoding gene with ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. Constitutive overexpression of B1 in awned wheat produced an awnletted phenotype with pleiotropic effects on plant height and fertility. Transcriptome analysis of B1 overexpression plants suggests a role as transcriptional repressor, putatively targeting pathways involved in cell proliferation. Haplotype analysis revealed a conserved B1 coding region with proximal polymorphisms and supported the contention that B1 is mainly responsible for awnletted wheats globally. B1, predominantly responsible for awn inhibition in wheat, encodes a C2H2 zinc finger protein with EAR motifs which putatively functions as a transcriptional repressor.


Assuntos
Genes Dominantes , Loci Gênicos , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Triticum/anatomia & histologia , Triticum/genética , Dedos de Zinco , Motivos de Aminoácidos , Pão , Proliferação de Células/genética , Mapeamento Cromossômico , Segregação de Cromossomos/genética , Regulação da Expressão Gênica de Plantas , Pleiotropia Genética , Haplótipos/genética , Ácidos Indolacéticos/metabolismo , Família Multigênica , Mutação/genética , Fases de Leitura Aberta/genética , Desenvolvimento Vegetal/genética , Polimorfismo Genético
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