Conditional degradation of SDE2 by the Arg/N-End rule pathway regulates stress response at replication forks

Abstract

Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2 Ct , the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2 Ct constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97 UFD1-NPL4 segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2 Ct degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2 Ct , fail to induce RPA phosphorylation and single-stranded DNA formation, leading to defects in PCNA-dependent DNA damage bypass and stalled fork recovery. Together, our study elucidates a previously unappreciated axis connecting the Arg/N-end rule and the p97-mediated proteolysis with the replication stress response, working together to preserve replication fork integrity.

Proteome dynamics analysis identifies functional roles of SDE2 and hypoxia in DNA damage response in prostate cancer cells

“…pcDNA-SDE2-Flag, pcDNA-SDE2-GG/AA-Flag and pcDNA-SDE2-K/V-Flag were plasmids used in our previous studies ( 22 , 26 ). Plasmid transfection was performed with jetPRIME ® transfection reagents.…”

“…Plasmid transfection was performed with jetPRIME ® transfection reagents. Sequences for control siRNA and siRNAs targeting SDE2, CDT2, UBR1 and UBR2 were synthesized by Dharmacon (Lafayette, CO) based on previous publications ( 22 , 26–27 ). HIF1α and HIF2α siRNAs were ordered from Qiagen (Germantown, MD).…”

SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

“…SDE2 Ct contains a conserved SDE2 domain of unknown function at its N-terminus, while its C-terminal SAP domain mediates the association of SDE2 Ct with chromatin. We further showed that chromatin-associated degradation of SDE2 Ct by Arg/N-end rule-p97 ATPase proteolytic pathway is necessary for propagating the signaling of the DNA replication stress response at RPA-coated stalled forks in response to UVC damage 35 . These findings indicate that the dynamic control of SDE2 protein levels may modulate protein complexes and their activities at stressed forks.…”

“…Although the exact nature of the FPC within a reversed fork remains elusive, it may act upon its alternative role of restricting nuclease activities to stabilize forks and suppress extensive generation of ssDNA intermediates, which is distinct from its canonical role coupling helicase-polymerase movement during unstressed conditions. We have previously shown that damagedependent degradation of SDE2 is required for the acute response to DNA replication stress 35 .…”

“…pcDNA3-SDE2-Flag and its mutants were previously described 34,35 . pcDNA4-Flag-TIMELESSmyc-6xHis and pcDNA3-Flag-TIPIN were a gift from Aziz Sancar (Addgene plasmids #22887 and #22889).…”