We have shown that the phosphothreonine binding motif of the FHA domain of Fkh1 plays a critical role in the regulation of donor preference (). A strong physical association between the FHAFkh1 domain bound at the RE region and MAT is readily seen, but only after a DSB is induced. This interaction is independent of the presence of an adjacent homologous HML donor (). Conversely, the region surrounding RE can be phosphorylated by Mec1 and Tel1 kinases only after DSB induction in MATa but not in MATα strains (), again suggesting that these regions can come into physical contact when there is a DSB at MAT and RE is active.
RE's activity does not depend on any of the special features of MAT
switching such as HML
or HO cleavage 
. Consequently RE is able to improve the use of an ectopic donor in repairing a DSB on a different chromosome. Normally, a DSB will be preferentially repaired by a donor on the same chromosome in competition with an ectopic donor, but if the ectopic donor is located near RE, more than half of gene conversions use the interchromosomal donor (). Although our data and those from others show that HML
is not constitutively much closer to MATa
is (i.e. in the absence of HO cleavage) 
, the data we present here suggest that such a reorganization will occur after a DSB is created.
Taken together, our data suggest a simple model for RE action (). After the induction of a DSB, casein kinase II and possibly other kinases modify some proteins bound near the DSB. These modifications, most likely phosphothreonines, are clustered near the DSB and can be bound by FHAFkh1
domains tethered at RE. This binding effectively tethers HML
within about 20 kb of the DSB whereas HMR
remains 100 kb away. Thermodynamic considerations argue that this close proximity is sufficient to explain why HML
should be used 90% of the time for DSB repair in MATa
. This model also explains how RE can act over a long region of the left arm of chromosome III 
, although with diminishing effect 
, by this tethering mechanism.
The model we propose argues that RE should be portable and able to stimulate the use of any homologous donor in a DSB repair mechanism. Our previous work has shown that RE is portable, as it is able to activate HML
use when both are inserted on chromosome V 
. Moreover, if a copy of RE is inserted near HMR
in a MATa
strain that also has RE near HML
, then HMR
usage is increased to about 50% (E.C., S.-Y. Tay and J.E.H., unpublished). The ectopic recombination experiment presented here shows that RE can act efficiently on non-MAT
sequences for DSB repair ().
We note that we have previously shown that RE could stimulate spontaneous recombination between leu2
heteroalleles when one of them was located close to the RE 
. The results we report here suggest that a large proportion of spontaneous recombination events may be triggered by DSBs or that the same phosphorylated protein attracting the attention of RE during DSB repair also accumulates at the lesions that stimulate spontaneous recombination.
At present, we have not yet identified the phosphothreonine target for the FHA domain of Fkh1. We have ruled out a number of candidates, including γ-H2AX, N-terminal tails of histones H3 and H4, as well as Mre11 and Sae2, two proteins involved in DSB end-binding and initiating 5′ to 3′ resection (C.-S. L., J.E.H., unpublished observations). Studies using peptide libraries and immunoprecipitation of the FHAFkh1 domain after DSB induction are underway.
Aparicio group has recently made the intriguing finding that Fkh1 and Fkh2 proteins play a key role in the activation and clustering of early origins of replication in budding yeast 
. This regulation involves a cis-acting association of these two forkhead proteins with proteins at origins. It will be interesting to ask if the FHA domain of Fkh1 plays an important role in this regulation.
Another important finding emerging from our work is that two DNA damage checkpoint kinases, Mec1/ATR and Tel1/ATM, can act to phosphorylate distant DNA sequences when they are tethered in the vicinity of the DSB. As shown in , the γ-H2AX modification spreads around the RE region, but with significantly delayed kinetics compared with the modification around MAT
, consistent with the idea that RE has to first recognize and bind to phosphorylated residues in the vicinity of the DSB at MAT
. How these checkpoint kinases act on their target sequences is not yet firmly established. Mammalian ATM has been shown to be activated by intermolecular autophosphorylation and dimer exchange, which would suggest that activated ATM would initially form a “cloud” of activated kinases around the site where the kinases were associated with the DSB ends 
. In the case of Tel1/ATM, the association with the DSB is via its association with the MRX/MRN proteins 
; in the case of Mec1/ATR, by its association its partner protein Ddc2/ATRIP with RPA bound to ssDNA at the resected DSB end 
. In budding yeast, the spreading of γ-H2AX from the DSB site is consistent with that the tethered kinases interact with phosphorylating histones on the adjacent chromosomal segment in a manner, which is similar to the contact of chromosomal regions as measured in chromosome conformation capture experiments 
. Spreading of γ-H2AX further along the chromosome occurs more slowly and apparently depends on the continuing 5′ to 3′ resection of the DSB ends, generating ssDNA, as it depends only on Mec1 
. Here we show that histones in another distant chromosomal region, brought into proximity with the DSB by RE, can also be efficiently phosphorylated – and by both Mec1 and Tel1. This result is different from the slow addition of γ-H2AX to regions further from the DSB, which depends on continuing 5′ to 3′ resection of the DSB ends and can only be performed by Mec1 
. We have also observed γ-H2AX spreading onto a different chromosome during the ectopic recombinational repair of a DSB, when these two regions are brought together by Rad51-mediated strand invasion (K.L. and J.E.H., unpublished observations).