Biophysical analysis of diffusion controlled processes in the budding yeast nucleus

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.