RESUMO
Ubiquitination controls numerous cellular processes, and its deregulation is associated with many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNA Pol I.
Assuntos
RNA Polimerase I , Ubiquitina , Sequência de Aminoácidos , RNA Polimerase I/metabolismo , Proteômica , Proteínas de Ciclo Celular/metabolismo , RNA , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.
Assuntos
Proteínas de Ciclo Celular/genética , Complexos Multiproteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Fenômenos Biofísicos , Proteínas de Ciclo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , Proteínas de Ligação a DNA/genética , Humanos , Interfase/genética , Mitose/genética , Complexos Multiproteicos/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Sumoilação/genéticaRESUMO
The Smc5/6 complex plays essential roles in chromosome segregation and repair, by promoting disjunction of sister chromatids. The core of the complex is constituted by an heterodimer of Structural Maintenance of Chromosomes (SMC) proteins that use ATP hydrolysis to dynamically associate with and organize chromosomes. In addition, the Smc5/6 complex contains six non-SMC subunits. Remarkably, and differently to other SMC complexes, the Nse1 and Nse2 subunits contain RING-type domains typically found in E3 ligases, pointing to the capacity to regulate other proteins and complexes through ubiquitin-like modifiers. Nse2 codes for a C-terminal SP-RING domain with SUMO ligase activity, assisting Smc5/6 functions in chromosome segregation through sumoylation of several chromosome-associated proteins. Nse1 codes for a C-terminal NH-RING domain and, although it has been proposed to have ubiquitin ligase activity, no Smc5/6-dependent ubiquitylation target has been described to date. Here, we review the function of the two RING domains of the Smc5/6 complex in the broader context of SMC complexes as global chromosome organizers of the genome.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Trifosfato de Adenosina/química , Cromossomos/ultraestrutura , DNA/química , Dano ao DNA , Reparo do DNA , Humanos , Hidrólise , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe , SumoilaçãoRESUMO
Replication of a damaged DNA template can threaten the integrity of the genome, requiring the use of various mechanisms to tolerate DNA lesions. The Smc5/6 complex, together with the Nse2/Mms21 SUMO ligase, plays essential roles in genome stability through undefined tasks at damaged replication forks. Various subunits within the Smc5/6 complex are substrates of Nse2, but we currently do not know the role of these modifications. Here we show that sumoylation of Smc5 is targeted to its coiled-coil domain, is upregulated by replication fork damage, and participates in bypass of DNA lesions. smc5-KR mutant cells display defects in formation of sister chromatid junctions and higher translesion synthesis. Also, we provide evidence indicating that Smc5 sumoylation modulates Mph1-dependent fork regression, acting synergistically with other pathways to promote chromosome disjunction. We propose that sumoylation of Smc5 enhances physical remodeling of damaged forks, avoiding the use of a more mutagenic tolerance pathway.
