Biophysical analysis of diffusion controlled processes in the budding yeast nucleus

Abstract

The spatial organization of chromosomes and the dynamics of chromosome reorganization have been shown to be crucial for the regulation of gene expression and various aspects of DNA metabolism. However, the mechanisms that establish and maintain nuclear organization and coordinate changes to this organization are poorly understood. I used computer simulations based on random walk and polymer chain models to investigate the diffusion controlled behavior of chromosomes and extrachromosomal elements in the yeast nucleus. I also investigated the influence of fundamental geometrical and physical parameters on this behavior. Concretely, I analyzed the following systems: 1.) The distribution of intrachromosomal telomere-telomere distances in yeast and the effects of telomere anchoring: I could show that the intrachromosomal telomere-telomere distances of chromosomes 3 and 6 obtained using fluorescence microscopy measurements are shorter on average than predicted by the model for the respective chromosomes in free solution in the nucleus, suggesting additional constraints. Furthermore, telomeric anchoring leads to a further increase in the average distance and can therefore not be directly responsible for the close juxtaposition. 2.) The influence of nuclear geometry on the diffusion of a plasmid during nuclear division: In budding yeast, autonomously replicating sequence ARS plasmids show a strong tendency to segregate to the mother cell at mitosis. I showed that the geometric shape of the dividing nucleus and the limited length of mitosis impose a severe barrier on passive diffusion into the daughter nucleus, explaining the asymmetry in plasmid distribution. In collaboration with a colleague, I could show experimentally and theoretically that tethering of ARS plasmids to the inner nuclear membrane can increase the efficiency of plasmid partitioning. Our results suggest that the asymmetric morphology of mitosis could potentially contribute to rejuvenation by imposing physical constraints on the diffusion of damaged material into the daughter. 3.) The influence of nuclear organization and specifically telomeric anchoring on the search for a template during homologous recombination: DNA double-strand breaks are the most deleterious DNA lesions. Homologous recombination uses a homologous template to repair a double-strand break accurately and is very efficient in yeast. However, the process by which the break and template site find each other within the vast quantity of non-homologous DNA is not well understood. We have developed a combined experimental and theoretical approach to study homology search and its relationship to nuclear organization in yeast. I introduce our strategy and present some first theoretical results that prove the concept of the approach. Within the ongoing work on homology search in yeast, I am going to investigate the important question of the influence of locus mobility on nuclear processes in yeast.