Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Abstract

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G 1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2 Rad6 and UBC3 Cdc34 . UBC2 Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3 Cdc34 goes through the Skp1͞Cdc53͞F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

DNA Damage Response-mediated Degradation of Ho Endonuclease via the Ubiquitin System Involves Its Nuclear Export

“…Here we demonstrate a direct interaction between Ufo1 and its substrate, Ho, and with the SCF subunits, Skp1 and Cdc53, and map these interactions to specific domains of the Ufo1 protein. Substrates degraded by the SCF are usually phosphorylated (8) and we identified a PEST sequence in Ho that when deleted led to stabilization of the protein (6). We show that phosphorylation of Ho is essential for its interaction with Ufo1 and that, in a ⌬mec1 mutant of the DDR, Ho is not phosphorylated and cannot bind Ufo1.…”

“…Ho degradation involves two E2s, UBC2 Rad6 and UBC3 Cdc34 (6). UBC3 Cdc34 ubiquitylates substrates as part of the Skp1-Cdc53-F-box receptor (SCF) E3 ubiquitin ligase complex (8,9) and Ho is stabilized in mutants of Skp1 and Cdc53 and also in a deletion of the putative F-box coding gene ORF YML088w (6). The SCF mediates the ubiquitylation of substrates whose degradation is necessary for cell cycle progression at the G 1 /S transition.…”

“…In contrast, repair of the Ho DSB by gene conversion leads to insertion of a new MAT allele and resurrects the Ho cognate site. We have shown that Ho endonuclease activity is confined to a narrow time window and that in addition to tightly regulated transcription of HO, the protein is degraded via the ubiquitin-26 S proteasome system with a short half-life of about 8 min (6). Ubiquitylation of proteins involves a cascade of enzymes: ubiquitin is activated by an ubiquitin-activating enzyme (E1) that transfers the activated ubiquitin to an ubiquitin-conjugating enzyme (E2/UBC).…”

“…Functions of the DNA damage response (DDR) MEC1, RAD9, and CHK1 are essential for degradation of Ho (6). The DDR is a network of interacting pathways for genome surveillance that leads to cell cycle arrest in response to DNA damage or to stalled replication and ensures that replication and chromosome segregation are completed with high fidelity (15,16).…”

“…Specificity of substrate ubiquitylation is mediated by an ubiquitin ligase (E3) that binds both the E2 and the substrate and mediates formation of the isopeptide bond between the terminal residue of ubiquitin and the substrate (7). Ho degradation involves two E2s, UBC2 Rad6 and UBC3 Cdc34 (6). UBC3 Cdc34 ubiquitylates substrates as part of the Skp1-Cdc53-F-box receptor (SCF) E3 ubiquitin ligase complex (8,9) and Ho is stabilized in mutants of Skp1 and Cdc53 and also in a deletion of the putative F-box coding gene ORF YML088w (6).…”