2D Gel Electrophoresis to Detect DNA Replication and Recombination Intermediates in Budding Yeast

Zardoni, Luca; Nardini, Eleonora; Liberi, Giordano

doi:10.1007/978-1-0716-0323-9_4

Cited by 4 publications

(1 citation statement)

References 27 publications

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“…Purification of DNA intermediates and 2D gel electrophoresis procedure were carried out as described in ( 55 ) using yeast strains synchronized in G1-phase of the cell cycle and released into 0.2 M hydroxyurea (HU). Briefly, yeast cells were exposed to in vivo psoralen cross-linking and replication intermediates were purified in the presence of CTAB.…”

Section: Methodsmentioning

confidence: 99%

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Zardoni

Nardini

Brambati

et al. 2021

Nucleic Acids Research

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Uncoordinated clashes between replication forks and transcription cause replication stress and genome instability, which are hallmarks of cancer and neurodegeneration. Here, we investigate the outcomes of head-on replication-transcription collisions, using as a model system budding yeast mutants for the helicase Sen1, the ortholog of human Senataxin. We found that RNA Polymerase II accumulates together with RNA:DNA hybrids at sites of head-on collisions. The replication fork and RNA Polymerase II are both arrested during the clash, leading to DNA damage and, in the long run, the inhibition of gene expression. The inactivation of RNA Polymerase II elongation factors, such as the HMG-like protein Spt2 and the DISF and PAF complexes, but not alterations in chromatin structure, allows replication fork progression through transcribed regions. Attenuation of RNA Polymerase II elongation rescues RNA:DNA hybrid accumulation and DNA damage sensitivity caused by the absence of Sen1, but not of RNase H proteins, suggesting that such enzymes counteract toxic RNA:DNA hybrids at different stages of the cell cycle with Sen1 mainly acting in replication. We suggest that the main obstacle to replication fork progression is the elongating RNA Polymerase II engaged in an R-loop, rather than RNA:DNA hybrids per se or hybrid-associated chromatin modifications.

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Section: Methodsmentioning

confidence: 99%

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Zardoni

Nardini

Brambati

et al. 2021

Nucleic Acids Research

Self Cite

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Yeast Proteins: Proteomics, Extraction, Modification, Functional Characterization, and Structure: A Review

Zhao,

Han,

Zhu

et al. 2024

J. Agric. Food Chem.

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Proteins are essential for human tissues and organs, and they require adequate intake for normal physiological functions. With a growing global population, protein demand rises annually. Traditional animal and plant protein sources rely heavily on land and water, making it difficult to meet the increasing demand. The high protein content of yeast and the complete range of amino acids in yeast proteins make it a high-quality source of supplemental protein. Screening of high-protein yeast strains using proteomics is essential to increase the value of yeast protein resources and to promote the yeast protein industry. However, current yeast extraction methods are mainly alkaline solubilization and acid precipitation; therefore, it is necessary to develop more efficient and environmentally friendly techniques. In addition, the functional properties of yeast proteins limit their application in the food industry. To improve these properties, methods must be selected to modify the secondary and tertiary structures of yeast proteins. This paper explores how proteomic analysis can be used to identify nutrient-rich yeast strains, compares the process of preparing yeast proteins, and investigates how modification methods affect the function and structure of yeast proteins. It provides a theoretical basis for solving the problem of inadequate protein intake in China and explores future prospects.

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Linear DNA-driven recombination in mammalian mitochondria

Fragkoulis,

Hangas,

Fekete

et al. 2024

Nucleic Acids Research

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Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

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2D Gel Electrophoresis to Detect DNA Replication and Recombination Intermediates in Budding Yeast

Cited by 4 publications

References 27 publications

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Yeast Proteins: Proteomics, Extraction, Modification, Functional Characterization, and Structure: A Review

Linear DNA-driven recombination in mammalian mitochondria

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