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

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

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

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

SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization. The fork protection complex (FPC), including the proteins TIMELESS and TIPIN, stabilizes the replisome to ensure unperturbed fork progression during DNA replication. Here the authors reveal that that SDE2, a PCNA-associated protein, plays an important role in maintaining active replication and protecting stalled forks by regulating the replication fork protection complex (FPC)