What regulates long-range DNA interactions that are necessary for recombination-mediated repair of DSBs remains a mystery. Even less clear is the mechanism through which a DSB preferentially recognizes one of two equivalent donor sites. Nonetheless this occurs in HO+
yeast cells once per cell cycle with remarkable fidelity. Previous studies have tried without success to identify mating-type-specific spatial clues that predispose one HM
locus and not the other to interact with the cleaved MAT
). Although neither found mating-type-specific juxtaposition, it was argued that Chr3L has a distinct mobility in MATa
α cells, which might favor exchange with MATa
). Our results confirm and extend these previous studies, showing that in G1
- and in S-phase cells prior to cleavage by HO, the radial subnuclear position and dynamics of HML
are largely mating type independent. Thus, it is unlikely that a predetermined spatial juxtaposition determines the directionality of the mating type switch.
On the other hand, we do find distinct mating-type specific characteristics that correlate with donor preference. First, yKu is enriched at the HML
α locus in MATa
cells compared to MAT
α cells. yKu binding to the silent mating type loci provides an alternate pathway to Sir1 for recruiting Sir4 to establish heterochromatin (41
). We confirm that in a sir4
deletion strain, yKu binding to HML
is abolished and as a consequence HML
relocates to the center of the nucleus. Intriguingly, upon DSB induction in a recombination-competent strain HML
α moves to a more internal position in MATa
, but not in MAT
α, cells. This movement is yKu dependent and thus correlates with mating-type-specific donor preference.
The yKu heterodimer is a highly conserved factor that is implicated in the repair of DSBs by nonhomologous end joining, a repair pathway that involves direct ligation of unresected DSB ends. Although mating type switching does not require nonhomologous end joining, being a form of HR, we did score an effect of yku70 deletion on the directionality of recombination: efficient mating type exchange is reduced by >20% in the absence of yKu. Our analysis of the effect of yKu70 on HML position and mobility may be able to explain this effect.
Through a careful quantitative analysis of locus position in a range of mutants, we attribute a unique position and behavior to Tel3L, which is dependent on the presence of HML
α. Whereas Tel3R and HMR
behave like other telomere proximal loci, HML
obeys other rules. First, perinuclear anchoring and dynamic constraint appear to be uncoupled, so that in the absence of yKu, the locus is shifted to the NE thanks to Sir4-Esc1 interactions, yet the constraints on its mobility are reduced. Indeed, in the absence of yKu70, the HML
locus moves readily along a radial trajectory but remains associated with the NE, a property earlier noted for Tel14L in a wt background (47
) or for the active GAL1
). The distinct behavior of HML
may be related to the general lack of recombination shown on the left arm of Chr3, an incompetence that can be overcome by the action of the RE (59
). Consistent with a model in which yKu and the RE work in parallel to ensure that recombination of HML
α with MATa
is favored, Coic et al. (10
) have recently proposed that the targets of RE function may be anchorage sites that tether Chr3L in MATa
cells. This is in full agreement with our quantitative microscopic analysis. We propose that the RE acts at the time of DSB cleavage to overcome a natural inaccessibility of HML
α, enabling fruitful recombination with MATa
We argue that the effect of yKu on the nuclear positioning of HML
contributes to the regulation of mating type exchange. In MATa
cells, yKu binds to HML
α in both the absence and presence of a DSB, while MAT
is bound only after cleavage (34
). We propose that high levels of yKu at HML
α favor recruitment of HML
α to the cleaved MATa
locus, which in turn would facilitate gene conversion thanks to the immediate juxtaposition of two homologous loci. Our observation that the amount of yKu binding to HML
is mating type specific reinforces the idea of an unexpected role for the yKu complex in donor choice. Thus, yKu may both directly facilitate long-range interactions of sites to which it binds, as well as establish a nuclear organization conducive to selective sequence exchange.
In the absence of yKu, HML is more efficiently sequestered at the nuclear periphery and this correlates with less efficient switching. This suggests that the tight perinuclear anchoring of HML inhibits its recruitment to the MAT locus during gene conversion. However, since the perinuclear anchoring of HMRa does not interfere with its recombination with MATα, we propose that the function of the RE is partially impaired when HML is sequestered at the NE (i.e., in the yku70 mutant). To reconcile this, we propose that HMRa and HMLα associate with distinct subnuclear anchorage sites, which affect the gene conversion event differently. This is supported by the fact that HMR position is not affected by yku70 deletions. Our results further imply that the role or complex formed by yKu at HML is not functionally equivalent to telomere-associated yKu. This is not unexpected, given the multiplicity of functions ascribed to the yKu heterodimer.
We have recently demonstrated that the probability with which two chromosomal loci interact is influenced by nuclear geometry and is reduced when the loci are tethered at the periphery (16
). Consistent with the notion that peripheral sequestration negatively affects HML-MAT
interaction, computer-driven simulations of spontaneous interaction between randomly moving loci argue that HML
tethering at an internal position leads to a twofold increase in the probability of spontaneous collision with MAT
, although contact was infrequent in all cases analyzed (L.G., K.B., and S.M.G., data not shown). This is particularly relevant since we find MAT
strongly enriched in the nuclear lumen even after induction of the DSB, as long as the strain is competent for recombinational repair. Only when an irreparable DSB is induced at MAT
, i.e., cleavage in a strain bearing deletions of HML
, does MAT
become sequestered at the nuclear periphery (37
). These observations are consistent with enrichment of Rad52 foci in the nuclear lumen under a wide range of damage conditions (Fig. ).
The nuclear and chromosomal architecture in budding yeast seems to offer a compromise that ensures the efficient regulation of two different processes at the same locus: transcriptional repression and a competence for selective recombination. Given that mating type interconversion occurs every cell cycle, yeast has opted for only one architectural plan for the two cell types, and it must therefore regulate the recombination event not by global positioning, but by quickly and locally activating the appropriate donor. This may be ensured by mating-type-specific yKu binding at HML.
This simple but plausible model is reminiscent of an idea proposed for higher eukaryotic nuclear organization (reviewed in reference 35
). Namely, it was proposed that elements of nuclear structure and the stable juxtaposition of chromosomal domains provide a scaffold for these sites during transient assembly of functional complexes during active chromosomal repair and transcription processes. The dynamic association and tethering of factors involved in HR, such as Rad51, to nuclear matrix-like structures may facilitate the repair of DNA damage (36
). Moreover, BRCA2-dependent association of Rad51 with a nuclear matrix was correlated with the formation of Rad51 nuclear foci in response to DNA damage (52
). In conclusion, we have identified a mechanism through which the yeast nucleus can sequester a specific locus in a reversible manner, possibly releasing it upon DSB induction to promote interaction with an internal DSB. The identification of further mechanisms that confer spatial constraint on specific DNA loci may yield new insights into the control of genome stability.