One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeast Saccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans—at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

Interphase chromosomes in Saccharomyces cerevisiae are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB) at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the silent mating locus (HML) on chromosome III in wild–type and mutant yeast strains that contain altered chromosome-tethering interactions. In wild-type yeast cells we find that disruption of the telomere tether does not dramatically change the position of HML with respect to the SPB, in agreement with theoretical predictions. Alternatively, using a mutant strain with a synthetic tether that localizes an HML-proximal site to the nuclear envelope, we find a significant change in the SPB-HML distance, again as predicted by theory. Our study quantifies the importance of tethering at telomeres on the organization of interphase chromosomes in yeast, which has been shown to play a significant role in determining chromosome function such as gene expression and recombination.

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...

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3’ end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. When every 10 th base is mismatched between donor and recipient, BIR is 44% efficient compared to fully homologous sequences; but BIR still occurs about 10% when every 6th base is mismatched. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, but where they are clustered with spacings of every 6th bp. These different arrangements of uneven mismatch distribution were in general less efficient in recombination as templates with evenly distributed mismatches. A donor with all 10 mismatches clustered every 6th base at the 3’ invading end of the DSB was not impaired compared to the case where mismatches were clustered at the 5’ end. These data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but sequence-specific factors are also important. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3’ end. Mismatch incorporation depends on the 3’→ 5’ proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins. Surprisingly, the probability of a mismatch 27 nt from the 3’ end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ “chews back” the 3’ end of the invading strand without any mismatch-dependent cues from the strand invasion structure.

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (-H2AX) around a DNA double-strand break (DSB). In the budding yeast S. cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1 ATR or Tel1 ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by ChIP-qPCR in order to uncover the mechanisms by which Mec1 ATR and Tel1 ATM propagate histone modifications across chromatin. With either kinase, -H2AX spreads as far as ~50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans -at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a 3D diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.