Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage inSaccharomyces cerevisiae.
During Nucleotide Excision Repair (NER) in the yeastS. cerevisiae, ubiquitylation of Rad4 is carried out by the E3 ubiquitin ligase that includes Rad7-Elc1-Cul3 and is critical to optimal NER. Rad7 E3 activity targets Rad4 for degradation by the proteaseome but, in principle, could also trigger other DNA damage responses. We observed increased nuclear ubiquitin foci (Ub-RFP) formation inS. cerevisiaecontaining a Rad7 E3 ligase mutant (rad7SOCS) in response to DNA damage by benzo[a]pyrenediolepoxide (BPDE) in dividing cells. Immunoblots reveal that ubiquitin conjugates of Rpn10 and Dsk2 accumulate in greater abundance inrad7SOCScompared toRAD7in dividing cells in response to BPDE which makes Rpn10 and Dsk2 candidates for being the ubiquitylated species observed in our microscopy experiments. Microscopy analysis with strains containing Dsk2-GFP shows that Dsk2-GFP and Dsk2-GFP/Ub-RFP colocalized in nuclear foci form to an increased extent in arad7SOCSmutant background in dividing cells than in aRAD7wild-type strain. Further, Dsk2-GFP in therad7SOCSstrain formed more foci at the plasma membrane following BPDE treatment in dividing cells relative to strains containingRAD7or arad7Δdeletion mutant. In response to a different agent, UV irradiation, levels of ubiquitylated proteins were increased inrad7SOCSrelative toRAD7, and the proteasomal deubiquitylase subunit, Rpn11 was even monoubiquitylated in the absence of damaging agents. Together these data show that Rad7 E3 activity attenuates ubiquitylation of proteins regulating the shuttling of polyubiquitylated proteins to the proteasome (Dsk2 and Rpn10) and removal of ubiquitin chains just prior to degradation (Rpn11). Since Rad7 E3 ligase activity has been shown to increase ubiquitylation of its target proteins, yet our results show increased ubiquitylation in the absence of Rad7 E3, we suggest that Rad7 E3 action regulates ubiquitin ligase and deubiquitylase (DUB) activities that act on Rpn10, Dsk2 and Rpn11.
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA, USA.
Joseph M
JM
Benoun
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA, USA.
Danielle
D
Lalimar-Cortez
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA, USA.
Analila
A
Valencia
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA, USA.
Adriana
A
Granda
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA, USA.
Destaye M
DM
Moore
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA, USA.
Eric P
EP
Kelson
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA, USA.
Paula L
PL
Fischhaber
DNA Damage Response; Dsk2; Rad7; Rpn10; Rpn11; Ubiquitin