Chromosomes are folded into cells in a nonrandom fashion, with particular genetic loci occupying distinct spatial regions. This observation raises the question of whether the spatial organization of a chromosome governs its functions, such as recombination or transcription. We consider this general question in the specific context of mating-type switching in budding yeast, which is a model system for homologous recombination. Mating-type switching is induced by a DNA double-strand break (DSB) at the MAT locus on chromosome III, followed by homologous recombination between the cut MAT locus and one of two donor loci (HMLα and HMRa), located on the same chromosome. Previous studies have suggested that in MATa cells after the DSB is induced chromosome III undergoes refolding, which directs the MAT locus to recombine with HMLα. Here, we propose a quantitative model of mating-type switching predicated on the assumption of DSB-induced chromosome refolding, which also takes into account the previously measured stochastic dynamics and polymer nature of yeast chromosomes. Using quantitative fluorescence microscopy, we measure changes in the distance between the donor (HMLα) and MAT loci after the DSB and find agreement with the theory. Predictions of the theory also agree with measurements of changes in the use of HMLα as the donor, when we perturb the refolding of chromosome III. These results establish refolding of yeast chromosome III as a key driving force in MAT switching and provide an example of a cell regulating the spatial organization of its chromosome so as to direct homology search during recombination.chromosome organization | homologous recombination | random-walk polymers | quantitative fluorescent microscopy | statistical physics C hromosomes in bacteria and eukaryotic nuclei are folded in a nonrandom fashion, which leads to specific DNA sequences assuming narrowly distributed positions within the cell or the nucleus (1-4). An interesting question is, then, to what extent does this spatial organization of chromosomes determine their function? For example, differential gene expression during development and the maintenance of genomic integrity in response to damaging agents have both been linked to chromosome organization. In both cases the folded state of chromosomes determines which functionally related DNA sequences are spatially close to each other, which in turn enables their function (5-11). Furthermore, earlier theoretical studies addressing aspects of long-distance regulatory interactions between DNA sequences have suggested that formation of chromosomal loops can be used to modulate the frequency of these interactions (12, 13). Double-strand breaks (DSBs) are deleterious DNA lesions that can be repaired by homologous recombination. The repair process requires spatial proximity, of about 10 nm or so, of the region around the DSB and a homologous DNA sequence, which is located either on the same or on a different chromosome (14-17). Recent studies have revealed a role for the spatial organization of...