Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage.
The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M(r) complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex.
Andrews
Emily A
EA
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom.
Palecek
Jan
J
Sergeant
John
J
Taylor
Elaine
E
Lehmann
Alan R
AR
Watts
Felicity Z
FZ
D000595:Amino Acid Sequence; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002470:Cell Survival; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004791:Enzyme Inhibitors; C479823:Nse2 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C486628:Smc5 protein, S pombe; C486630:smc6 protein, S pombe; D006918:Hydroxyurea