Visualizing the spatiotemporal dynamics of DNA damage in budding yeast.

Fluorescence microscopy has enabled the analysis of both the spatial distribution of DNA damage and its dynamics during the DNA damage response (DDR). Three microscopic techniques can be used to study the spatiotemporal dynamics of DNA damage. In the first part we describe how we determine the position of DNA double-strand breaks (DSBs) relative to the nuclear envelope.

Chromosome conformation maps in fission yeast reveal cell cycle dependent sub nuclear structure.

Successful progression through the cell cycle requires spatial and temporal regulation of gene transcript levels and the number, positions and condensation levels of chromosomes. Here we present a high resolution survey of genome interactions in Schizosaccharomyces pombe using synchronized cells to investigate cell cycle dependent changes in genome organization and transcription. Cell cycle dependent interactions were captured between and within S.

Chromosome positioning and the clustering of functionally related loci in yeast is driven by chromosomal interactions.

In recent years there has been considerable and growing interest in the 3-dimensional organization of genomes. In this manuscript we present an integrated computational-molecular study that produces an ensemble of high-resolution 3-dimensional conformations of the budding yeast genome. The compaction, folding and spatial organization of the chromosomes was based on empirical data determined using proximity-based ligation.

Targeted INO80 enhances subnuclear chromatin movement and ectopic homologous recombination.

Chromatin in the interphase nucleus moves in a constrained random walk. Despite extensive study, the molecular causes of such movement and its impact on DNA-based reactions are unclear. Using high-precision live fluorescence microscopy in budding yeast, we quantified the movement of tagged chromosomal loci to which transcriptional activators or nucleosome remodeling complexes were targeted.

Mitochondrial-nuclear DNA interactions contribute to the regulation of nuclear transcript levels as part of the inter-organelle communication system.

Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions.

Nuclear geometry and rapid mitosis ensure asymmetric episome segregation in yeast.

Background: Asymmetric cell division drives the generation of differentiated cells and maintenance of stem cells. In budding yeast, autonomously replicating sequence (ARS) plasmids lacking centromere elements are asymmetrically segregated into the mother cell, where they are thought to contribute to cellular senescence. This phenomenon has been proposed to result from the active retention of plasmids through an interaction with nuclear pores.

Visualizing yeast chromosomes and nuclear architecture.

We describe here optimized protocols for tagging genomic DNA sequences with bacterial operator sites to enable visualization of specific loci in living budding yeast cells. Quantitative methods for the analysis of locus position relative to the nuclear center or nuclear pores, the analysis of chromatin dynamics and the relative position of tagged loci to other nuclear landmarks are described. Methods for accurate immunolocalization of nuclear proteins without loss of three-dimensional structure, in combination with fluorescence in situ hybridization, are also presented.

Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination.

Telomeres form the ends of linear chromosomes and protect these ends from being recognized as DNA double-strand breaks. Telomeric sequences are maintained in most cells by telomerase, a reverse transcriptase that adds TG-rich repeats to chromosome ends. In budding yeast, telomeres are organized in clusters at the nuclear periphery by interactions that depend on components of silent chromatin and the telomerase-binding factor yeast Ku (yKu).