RESUMO
Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Genoma Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Culina/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Mitose/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability.