While temperature-sensitive alleles of the SUMO pathway genes (ubc9-1
) show severe cell cycle defects, the requirement of SUMO for cell viability in budding yeast is largely unexplained. However, the persistently low levels of target modifications by SUMO is a likely indicator that sumoylated fractions of proteins have biological roles that are distinct from the non-sumoylated pools of the same proteins. Considering the enrichment of sumoylated proteins at some nuclear subdomains, it is plausible that the essential roles of SUMO include specific activities of sumoylated proteins at these subnuclear regions, such as the centromere 
and the nucleolus 
. In our previous and current work we showed that the SUMO conjugates became predominantly nucleolar when the removal of conjugates was compromised ( and 
). Furthermore, the population of cells that breaks through the metaphase arrest induced by SUMO E3 dysfunction is prone to nucleolar nondisjunction (), which might account for the rDNA loss in these triple E3-deficient cells (). Thus, nucleolar proteins, particularly ones functioning in rDNA chromatin, can be key targets of SUMO in the nucleus.
What essential rDNA functions might require SUMO? Three key factors are known to contribute to the stability of rDNA arrays: (1) silencing, i.e. suppression of sister chromatid recombination and ensuing formation of extrachromosomal rDNA 
; (2) amplification controls maintaining the optimal size of the rDNA array 
; and (3) proper rDNA segregation 
. Topoisomerases probably participate in all of these processes and, with respect to SUMO, our previous work suggests that lacking Top2p sumoylation can lead to rDNA defects, as the rDNA array stability was reduced to a similar degree in top2ΔC
, or siz1Δsiz2Δ
. Thus, we proposed a hypothesis 
that the top1Δ top2ΔC
double mutant essentially phenocopies the rDNA destabilization phenotype of more severe top1Δ top2
double mutants 
In the present work we made the first step in testing the more general idea that other sumoylation targets cooperate with sumoylated Top2p in maintaining rDNA stability. In the process we uncovered an important pathway controlled by Mms21p in the unchallenged cell cycle. Indeed, analysis of the mms21-CH top2ΔC double mutant showed that the combined deficiency in Top2p sumoylation and in Mms21p SUMO E3 function do lead to strong rDNA segregation defects (). These defects are exacerbated by the loss of Top1p activity or Top1p sumoylation in corresponding triple mutants (). While at this junction we cannot attribute the lethality of top1Δ top2ΔC mms21-CH either exclusively to the loss of sumoylation or solely to rDNA dysfunction, it does raise the possibility that Siz1/Siz2-sumoylated Top1p and Top2p cooperate with Mms21-sumoylated proteins in rDNA maintenance. Additionally considering that the mitotic sumoylation of cohesin and condensin largely depends on Mms21p (), and that there is a synthetic lethality between top1Δ, top2ΔC and smc2KR6 mutations, one can hypothesize that activities of sumoylated pools of condensin and cohesin are either redundant or cooperative with topoisomerases in rDNA.
More extensive experiments on the individual Mms21p substrates are needed to elucidate their molecular roles in this pathway. Top1p and Top2p are needed for both efficient transcription and DNA replication, and the Mms21p E3 activity is important to confront DNA replication-induced stress 
; therefore it is possible that due to strong rDNA transcription and its asymmetric replication the resolution of transcription and replication impediments in top1Δ top2ΔC
cells specifically requires Mms21p SUMO E3 activity. Although it remains to be tested at the molecular level, this idea is consistent with the fact that Top2p and Top1p co-localize with the Smc5p/Smc6p complex and replication/transcription landmarks genome-wide 
, particularly at rDNA (S. D., to be published elsewhere).
The putative cooperation between distinct sumoylation targets (being either different proteins or different E3-specific sumoylation sites) of Siz1p, Siz2p, and Mms21p in the nucleolus may extend beyond rDNA itself. For example, many proteins involved in ribosome biogenesis are found to be sumoylated 
; such “clustered” SUMO conjugations may be important for nucleolar integrity as well. In addition, we previously reported that condensin is enriched at the tRNA genes 
, which are transcribed by RNA Pol III. The Mms21-regulation of cohesin's and condensin's binding () to the 5S rRNA gene (also transcribed by Pol III) may point to a novel SUMO-controlled function common to Pol III genes, such as their recruitment to the nucleolus 
. Further work is needed to examine these possibilities.