Nse4 is essential for progression of the cell cycle
Under non-permissive conditions, conditional nse4 mutants arrest as large budded cells with a 2C nucleus at the bud neck and a short mitotic spindle. The 2C nucleus represents a genuine post-S-phase arrest, as replicative intermediates are not detected when chromosomes are resolved by PFGE. In addition, there is no chromosome breakage as we do not detect sheared DNA by PFGE. The phenotype of nse4ts mutants at non-permissive temperatures has the hallmarks of an arrest caused by activation of the DNA damage checkpoint. In budding yeast damage induced arrest occurs prior to anaphase but is often termed G2/M arrest.
Phosphorylation of the effector checkpoint kinase Rad53 is clearly detectable in arrested nse4ts cells. We demonstrated a requirement for an intact RAD24 checkpoint for the nse4ts cell cycle arrest. This in combination with our FACS and PFGE data, showing complete replication of DNA in arrested cells, suggests Rad53 activation in nse4ts cells is more likely a consequence of activation of the damage checkpoint and not the intra-S checkpoint. Rad53 phosphorylation infers that loss of Nse4 function leads to disturbance in chromosomal structure. Not surprisingly all four of our nse4ts mutants are hypersensitive to agents that generate aberrant DNA structures, namely stalled replicative intermediates generated by the replication inhibitor HU, and DNA damage caused by the methylation agent, MMS.
Physical interaction of Nse4 with components of the Smc5/Smc6 DNA repair complex
There are numerous complexes involved in DNA repair. Nse4 may play a role in the function of one of these, and we anticipated that two-hybrid screening would identify the complex. Use of Nse4 as bait reveals a strong interaction with Ydr288w, a protein of unknown function. As expected, the reverse screen using Ydr288w as bait, picks up Nse4 and a strong interaction with Nse1 (Non-Smc element 1) a component of a known DNA repair complex, Smc5/6 (Lehmann et al., 1995; Verkade et al., 1999). As is the case for nse4ts alleles, all mutants in the core Smc5/6 complex plus mutants in the known non-Smc elements, confer sensitivity to DNA damaging agents. This includes mutations in: S. cerevisiae SMC6/RHC18 and its S. pombe orthologue, rad18 (Lehmann et al., 1995; Onoda et al., 2004), the S. cerevisiae and S. pombe NSE1 orthologues (Fujioka et al., 2002; McDonald et al., 2003) and NSE2 orthologues (Prakash and Prakash, 1977; McDonald et al., 2003). Additionally, like nse4-2 ts cells, smc6 ts mutants also exhibit a mitotic instability phenotype (Onoda et al., 2004). Consistent with a nuclear role, GFP tagged Smc5, Smc6, Nse4, Ydr288w and Mms21 (the S. cerevisiae orthologue of S. pombe Nse2) all localize to the nucleus, as does Nse1 (Fujioka et al., 2002; Huh et al., 2003; McDonald et al., 2003).
The mutant phenotype data plus data from our two-hybrid screens suggest a role for Nse4 in Smc5/6 function, which was corroborated by functional interaction between Nse4 and members of the Smc5/6 complex. Growth arrest of nse4-1ts was suppressed by overexpression of SMC5; nse4-4ts growth arrest was suppressed by overexpression of NSE1. That neither SMC5 nor NSE1 overexpression rescued both mutants is intriguing, undoubtedly reflecting the different positions of the amino acid substitutions in the two nse4ts mutants and consequently the role these sites play in the function of the Smc5/6 holocomplex. Both nse4ts mutants were rescued by overexpression of YDR288w. A very recent publication has identified the S. pombe orthologue of Ydr288w as a component of the Smc5/6 complex; this S. pombe orthologue has been named Nse3 (Pebernard et al., 2004). This protein is essential in both budding and fission yeast. In S. pombe, Nse3 is necessary for mitotic chromosome segregation and cellular resistance to genotoxic agents. We have shown that all of these properties are shared by cells lacking Nse4, the S. cerevisiae binding partner for Ydr288w/Nse3. Furthermore, the interaction between Nse4 and Ydr288w/Nse3 is conserved in eukaryotes as the recently completed two-hybrid interaction map for Drosophila melanogaster reveals an interaction between the fly orthologues of these proteins (Giot et al., 2003). We propose renaming YDR288w as NSE3 (non-SMC element 3), in keeping with the name proposed for the S. pombe orthologue of this protein by Pebernard and coworkers. We have registered the name NSE3 as an alias for YDR288w, at the SGD.
Pebernard and coworkers point out that Nse3/Ydr288w is orthologous to the melanoma antigen (MAGE) family in humans. The function of MAGE has been obscure, until the work presented by Pebernard et al. proposed a role in the Smc5/6 DNA repair complex. Humans contain over 20 members of the MAGE family, though Drosophila and yeast contain one (Pebernard et al., 2004). In humans, MAGE has been implicated in cell cycle regulation and inhibition of apoptosis (Barker and Salehi, 2002).
Recent data from proteomic studies substantiates the role we suggest for Nse4 in Smc5/6 function. TAP tagged Nse3/Ydr288w copurifies with a complex containing Nse4, Smc5, Smc6, Nse1 and Mms21 (the S. cerevisiae orthologue of S. pombe Nse2) (Hazbun et al., 2003). Some of these proteins are undoubtedly the orthologues of the unidentified non-Smc proteins in S. pombe that bound to epitope tagged Smc6 (Fousteri and Lehman, 2000). Our two-hybrid data, plus two-hybrid and TAP-tag interaction data generated by Hazbun and coworkers, demonstrate that Nse4 and Nse3/Ydr288w are physically part of the Smc5/6 complex. We have also shown that Nse4 plays a functional role in this complex, as overexpression of Smc5/6 components suppresses the phenotype of nse4 conditional mutants. A composite schematic combining our two-hybrid data with two-hybrid and TAP-tag interaction data described elsewhere, plus our functional interaction data, is shown in Fig. 7B. Recent data on Rad62, the S. pombe orthologue of NSE4 are in agreement with our results, indicating a role in DNA repair. The S. pombe rad62-1 mutant is hypersensitive to genotoxic agents, and is defective in repair of double-strand breaks; in addition, Rad62 coimmunoprecipitates with epitope tagged Smc5 (Morikawa et al., 2004).
Our two-hybrid screen also revealed interactions between Nse3/Ydr288w and a putative membrane protein (YCL073c) a ubiquitin carboxyl-terminal hydrolase (Ubp9) and a component of a mitotic signalling network required for isotropic bud growth (Rei1). None of these proteins are involved in DNA repair or maintenance of higher-order chromosome structures, so functional interaction with Nse3/Ydr288w is not immediately apparent. Our two-hybrid screen using Nse4 as bait identified Nse3/Ydr288w as the only binding partner. This interaction was also revealed by a genome-wide two-hybrid screen, which also identified seven other binding partners (Uetz et al., 2000). Three of these, Hsc82, Sti1 and Cpr6, are components of the Hsp90 chaperosome (Pearl and Prodromou, 2000). This chaperosome is responsible for creating and maintaining the active conformation of key regulatory proteins, and may play a similar role in maintenance of Smc5/6 holocomplex activity. The remaining Nse4 interactors were a repressor of transcription (Tup1) a component of a MAP kinase cascade that responds to osmotic shock (Ssk2) and an enzyme that catalyses the first step of GMP biosynthesis (Imd2). A functional interaction between these proteins and Nse4 is not immediately apparent.
Overall, the function of SMC complexes is to establish the higher-order structure of chromosomes. Smc1/3 (cohesin) is the glue that ensures sister chromatids are maintained as a pair; Smc2/4 (condensin) mediates mitotic chromosome condensation (Hirano, 2000). Higher-order function of the Smc5/6 heterodimer remains poorly understood. Overall, it seems maintenance of higher-order structures is of importance to DNA repair and replication. Mutations in cohesin subunits lead to increased sensitivity to genotoxic agents (Tatebayashi et al., 1998; Walowsky et al., 1999). The same is true for condensin subunits (Aono et al., 2002; Chen et al., 2004). Studies in S. pombe indicate a role for Smc5/6 in homologous recombination based DNA repair (Lehman et al., 1995). A conditional mutant of S. cerevisiae SMC6 is unable to induce interchromosomal recombination in response to DNA damage (Onoda et al., 2004). However, both SMC5 and SMC6 (like NSE4) are essential for progression of the undisturbed cell cycle, in the absence of extrinsic agents that damage DNA. It has been suggested that the Smc5/6 complex holds together broken DNA molecules in the vicinity of double-strand breaks, so that repair by recombination is allowed to take place (Fousteri and Lehman, 2000). This is plausible given that occasional double-strand breaks occur during DNA replication (Muris et al., 1996). It is not surprising that a complex involved in maintaining higher-order chromosome structure plays a role in recombination. In human cells, Smc1/3 promotes repair of DNA lesions by homologous recombination (Jessberger et al., 1996). Indeed, Smc5/6 seems to facilitate various DNA repair processes, which would explain why S. pombe smc6 mutants are sensitive to a wide range of DNA damaging agents (Harvey et al., 2004). Furthermore mutants in smc6 are synthetically lethal with a mutation in DNA topoisomerase 2 (Verkade et al., 1999). All of this suggests that Smc5/6, like the Smc1/3 and Smc2/4 complexes, plays a role in chromatin organization. In cells treated with the genotoxic agents HU and MMS, nse4ts cells lose viability. It is not activation of a checkpoint that is lost here, because the Rad53 effector kinase is activated in the mutants, even at non-permissive temperatures. HU causes stalling of replication forks, which also occurs when S phase cells encounter DNA alkylated by MMS (Tercero et al., 2003). Cell death under these circumstances implies a role for stabilization of stalled forks by Nse4, and by the Smc5/6 complex as a whole. The association of human Smc5/6 with DNA during interphase, and its exclusion from DNA during mitosis, is in agreement with this (Taylor et al., 2001).
Unlike mutants in smc5 and smc6, nse4 mutants arrest at a discrete stage in the cell cycle
In S. pombe, cells deleted for either Smc5 or Smc6 display a range of lethal terminal phenotypes such as elongated cells, many with several septa with a single nucleus, or a dispersed distribution of DNA or a cut phenotype. Chromosome missegregation has also been reported for a conditional smc6 mutant in S. cerevisiae (Onoda et al., 2004). Similar phenotypes are exhibited by the ts mutants in the non-Smc subunits, Nse1 and Nse2 (Fujioka et al., 2002; McDonald et al., 2003). Our nse4ts mutants, however, display a terminal phenotype that is far less extreme, namely a growth arrest prior to onset of anaphase. Moreover, this arrest is not lethal, even when the cells are incubated at non-permissive conditions for 3 days. Also, the nucleus is not fragmented at all, and is arrested at a stage prior to anaphase. In FACS analysis of smc6 ts mutants, the G2 peak becomes broader and flatter after 10 h at non-permissive conditions, reflecting the chromosome missegregation that occurs (Onoda et al., 2004). This does not occur in nse4ts mutants, the G2 peak persisting for at least 24 h. If the Smc5/6 complex repairs a distinct type of genomic lesion, characterization of this lesion will be easier in nse4ts mutants. Moreover, the catastrophic terminal phenotype of Smc5/6 mutants implies a collapse in the structure of the complex itself, leading to amorphous distribution of nuclear DNA and ‘cut’ morphology. This is not surprising because Smc5/6 forms the core of the complex. In nse4ts mutants, loss of function does not result in large-scale chromosomal aberrations, implying the central role of the Smc5/6 core remains intact. The nse4ts mutant phenotype may represent a ‘snapshot’ of the Smc5/6 mechanism of action.
In S. pombe, Smc5/6 functions in tandem with Rad60 (Morishita et al., 2002; Boddy et al., 2003). The budding yeast orthologue of Rad60 is Esc2, named for its role in chromatin silencing (Dhillon and Kamakaka, 2000). We have noticed that we are unable to synchronize the most severe nse4ts mutant (nse4-2 ts) with α-factor. This could be because of a silencing defect, giving rise to simultaneous expression of MATα and MATa genes. It is possible that Nse4 is involved in repair of damage, such as double-strand breaks, perhaps through assembly of newly repaired DNA into chromatin. We note that mutants in components of the Smc5/6 complexes exhibit deficiencies in recombination. PFGE of these cells would reveal sheared DNA. However, we do not see sheared DNA in arrested nse4ts cells. This is why we suggest a possible role for Nse4 in chromatin assembly post DNA repair. Moreover, S. cerevisiae which lack Asf1, a protein that mediates chromatin assembly, are – like nse4ts cells – sensitive to MMS and HU, and cannot be synchronized with α-factor (Tyler et al., 1999; Hu et al., 2001). The next key issue to be addressed is the exact nature of the chromosomal aberration that exists in arrested nse4ts cells. We propose to do this by investigating genetic and biochemical interaction between Nse4 and proteins involved in recombination repair and chromatin assembly.