During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks.
The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.
Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Denise E
DE
Bustard
Demis
D
Menolfi
Kristian
K
Jeppsson
Lindsay G
LG
Ball
Sidney Carter
SC
Dewey
Katsuhiko
K
Shirahige
Camilla
C
Sjögren
Dana
D
Branzei
Jennifer A
JA
Cobb
D000483:Alleles; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000737:chemistry; D006918:Hydroxyurea / Q000494:pharmacology; D009154:Mutation; D010641:Phenotype; D025842:SUMO-1 Protein / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological / Q000187:drug effects; D058207:Sumoylation / Q000187:drug effects; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; C575002:NSE5 protein, S cerevisiae; C486627:SMC5 protein, S cerevisiae; C486631:SMC6 protein, S cerevisiae; D025842:SUMO-1 Protein; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases; D006918:Hydroxyurea