Failure of the HR pathways underlies many human diseases including cancer and can cause birth defects through aberrant meiotic chromosome segregation. Here, we have identified a novel function for Nse5–Nse6 of the Smc5–Smc6 complex in processing mitotic and meiotic HR intermediates. Genetic and physical analyses indicate that nse6Δ mutants accumulate JMs resembling the HJs that accumulate in mus81Δ during meiosis. Thus, to our knowledge, Nse5–Nse6 is only the second factor in fission yeast required for the endonucleolytic processing of HJs, besides Mus81–Eme1, which has the catalytically active site for HJ resolution.
The key data supporting a function for Nse5–Nse6 in the resolution of JMs are as follows: (i) like mus81Δ
mutants form few viable spores (), due to the failure of chromosome segregation; (ii) like mus81Δ
is epistatic to nse6Δ
in meiosis, demonstrating that Nse5–Nse6 acts after DSB formation (); (iii) the double nse6Δ mus81Δ
mutant also forms few viable spores and is suppressed, at least partially, by rec12Δ
, indicating that Mus81 and Nse6 act in the same or closely related steps of meiosis (); (iv) the rad51Δ dmc1Δ
combination is epistatic to nse6Δ
, demonstrating that Nse5–Nse6 acts after the formation of JMs (); (v) JMs accumulate in meiotic nse6Δ
and nse6Δ mus81Δ
cells, and these JMs are temporally, genetically and electrophoretically indistinguishable from the HJs that accumulate in mus81Δ
cells ( and , (8
)); (vi) at least some of these JMs are sensitive to E. coli
RuvC HJ resolvase () and to RusA HJ resolvase, because, as for mus81Δ
), expression of RusA partially rescues the meiotic defects of nse6Δ
cells (C and D) and (vii) crossovers are modestly reduced in nse6Δ
cells, indicating that Nse5–Nse6 is important but not absolutely essential for HJ resolution, which requires Mus81–Eme1 ().
In addition to HJ resolution, Nse5–Nse6 appears to have a second role because (i) the double nse6Δ mus81Δ mutant grows more slowly and is more CPT sensitive than either single mutant (D); (ii) the double mutant is not quite as well suppressed to CPT resistance by expression of RusA as the single mutants (D); (iii) unlike mus81Δ, nse6Δ is defective for mitotic DSB repair, as is rad51Δ (); (iv) crossovers are formed, although at reduced level compared to wild-type, in nse6Δ but not in mus81Δ or nse6Δ mus81Δ mutants () and (v) the JMs that accumulate in nse6Δ and nse6Δ mus81Δ meiotic cells are at least partially sensitive to S1 nuclease, whereas those that accumulate in mus81Δ meiotic cells are not (A and B), suggesting that Mus81–Eme1 and Nse6 have distinct, and overlapping, roles.
To reconcile these observations, we propose that in meiotic cells Nse5–Nse6 stimulates the resolution of HJs by Mus81–Eme1 and the resolution of other structures, such as hemicatenanes, which may arise during mitotic growth or during meiosis in the absence of Mus81–Eme1. These structures may arise during mitotic replication, as suggested by others (45
), thereby partially accounting for the mitotic phenotypes of nse6Δ
mutants. The structure of the JMs that accumulate in nse6Δ
and nse6Δ mus81Δ
mutants is not entirely clear, although those that accumulate in mus81Δ
mutants are clearly single HJs (8
). The JMs that accumulate in nse6Δ
mutants are sensitive to both S1 nuclease and RuvC HJ resolvase, whereas those that accumulate in mus81Δ
mutants are sensitive to RuvC and those in nse6Δ mus81Δ
mutants are sensitive to S1 nuclease (A and B). The stimulation of Mus81 HJ resolvase activity by the Nse5–Nse6 complex may be direct or indirect.
We propose that in nse6Δ
mutants, Mus81–Eme1 slowly resolves HJs while some are converted into another structure and that in nse6Δ mus81Δ
mutants, most or all of the HJs are converted into this structure. This proposal is consistent with the slight reduction in meiotic crossover frequency, among the few viable spores that arise, in nse6Δ
mutants but strong reduction in mus81Δ
mutants (A (8
)); with the slight reduction in total crossover DNA in nse6Δ
mutants but strong reduction in mus81Δ
and nse6Δ mus81Δ
mutants (B (8
)); and with the suppression of nse6Δ
, but not nse6Δ mus81Δ
, by expression of the RusA HJ resolvase (C and D).
The resistance to branch migration of the IH JMs in nse6Δ
mutants suggests that these JMs, between heterozygous restriction sites, are single HJs with recombinant length strands (B). This is because either double HJs, which have parental length strands, or hemicatenanes with either recombinant or parental length strands should dissociate into separate duplexes on heating (e.g.
)). IS JMs would dissociate in any case, as observed. IH and IS JMs may differ in structure, but their low level has precluded our determining their sensitivity to S1 nuclease and RuvC. Although the S1 nuclease sensitivity is consistent with some JMs being hemicatenanes, to our knowledge, this structure has not been clearly demonstrated to arise in cells, for example by electron microscopy or comparison with synthetic DNA molecules. Further investigation is required to establish the structure of the population of non-HJ containing JMs in the absence of Nse6.
As Nse5–Nse6 acts as part of the Smc5–Smc6 complex, which has multiple roles in chromosome metabolism (44
), it is no surprise that Nse5–Nse6 has multiple roles. One tempting hypothesis is that during meiosis, the primary role of Nse5–Nse6 is to stimulate the Mus81–Eme1 HJ resolvase and that during mitotic growth it plays both this and a second role. During both stages of the life cycle, expression of RusA suppresses at least partially the nse6Δ
phenotype (D and C and D), indicating that HJ resolution is stimulated by Nse5–Nse6 in both stages. The second role of Nse5–Nse6 might be to regulate any of the multiple functions of the Smc5–Smc6 complex (44
). Further investigation is required to elucidate this function, but the requirement for Rad51 and Nse6 but not Mus81–Eme1 in mitotic DSB repair () suggests that Nse5–Nse6 is also important for the synapsis phase of mitotic DSB repair.
Although the Nse1 and Nse2 E3 ligase activities, like Nse5–Nse6, facilitate mitotic DNA repair (79–81
), neither of these E3 ligases is required for meiotic nuclear division and recombination [(82
) our unpublished data]. Thus, although the mitotic functions of Nse1, Nse2 and Nse5–Nse6 have not been dissected, in meiosis, Nse5–Nse6 acts independently of posttranslational modifications catalyzed by Nse1 and Nse2. Nevertheless, on the basis of our previous analyses showing that hypomorphic mutants of the essential Smc5–Smc6 subunits exhibit catastrophic meioses (83
), we propose that Nse5–Nse6 acts in conjunction with the Smc5–Smc6 holocomplex to execute its meiotic HR role.
Budding yeast smc5–smc6
mutations also disrupt meiotic nuclear division (84
), but the underlying defects appear strikingly different from those of nse6Δ
fission yeast. Key differences are that a spo11
homolog) mutation is not epistatic to an smc5–smc6
mutation, and crossovers are not affected in smc6
temperature-sensitive (Ts) mutant cells (84
). Thus, the authors concluded that the crucial role of Smc5–Smc6 is executed during premeiotic S phase in budding yeast and not after the initiation of meiotic recombination. It is unclear whether this reflects real differences in the functions of Smc5–Smc6 between species or if the unavoidable disruption of both the essential and repair roles in smc6
(Ts) budding yeast masks functions analogous to those of fission yeast Nse5–Nse6.
HJ resolution must be carefully controlled, both during mitotic growth and in meiosis. When a DNA strand lesion blocks mitotic replication, the fork can regress and form a structure whose center is identical to that of an HJ. Were it resolved by Mus81–Eme1, a DSB would be formed and require further processing to allow completion of replication. Alternatively, the regressed fork can migrate back to the original position and allow immediate continuation of replication. Thus, Mus81–Eme1 may be kept inactive during this time. If strand exchange between sisters or homologs forms an HJ that is present at the time of mitosis, HJ resolution would appear to be the most expedient means to allow chromosome segregation, and Mus81–Eme1 may be activated at this time [e.g. (19
)]. Similarly, during meiosis, many dozens of HJs must arise to account for the ~45 crossovers in a fission yeast cell (85
), and Mus81–Eme1 is clearly highly active in meiotic cells. We surmise that Nse5–Nse6, likely as part of the Smc5–Smc6 complex, regulates directly or indirectly the activity of Mus81–Eme1 allowing it to function at the proper time and place to maintain chromosome integrity and cell viability.