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

DNA and RNA Cleavage Complexes and Repair Pathway for TOP3B RNA- and DNA-Protein Crosslinks

The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a self-trapping mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2

Precision Oncology with Drugs Targeting the Replication Stress, ATR, and Schlafen 11

Precision medicine aims to implement strategies based on the molecular features of tumors and optimized drug delivery to improve cancer diagnosis and treatment. DNA replication is a logical approach because it can be targeted by a broad range of anticancer drugs that are both clinically approved and in development. These drugs increase deleterious replication stress (RepStress); however, how to selectively target and identify the tumors with specific molecular characteristics are unmet clinical needs. Here, we provide background information on the molecular processes of DNA replication and its checkpoints, and discuss how to target replication, checkpoint, and repair pathways with ATR inhibitors and exploit Schlafen 11 (SLFN11) as a predictive biomarker

Replication-associated formation and repair of human topoisomerase IIIα cleavage complexes

This study provides evidence that human topoisomerase IIIα (TOP3A) is closely associated with active replisomes. The authors uncover TOP3A DNA cleavage complexes (TOP3Accs) repair mechanisms to ensure normal DNA replication and genome integrity

Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway.

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity

PCNA-Dependent Cleavage and Degradation of SDE2 Regulates Response to Replication Stress

<div><p>Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4^CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4^CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function.</p></div

A proposed model for the regulation of SDE2 by PCNA-dependent cleavage and degradation.

<p><b>(A)</b> Targeting of SDE2 to PCNA-associated replication forks via the N-terminal UBL containing a PIP box leads to the cleavage of SDE2 at the diglycine motif. DUB activity is required for its cleavage. <b>(B)</b> The cleaved C-terminal SDE2 functions as a negative regulator of damage-inducible RAD18-dependent PCNA monoubiquitination. The SDE2 domain is required for this process. <b>(C)</b> Degradation of the cleaved N-terminal and C-terminal SDE2 products by CRL4^CDT2 allows timely S phase progression and promotes replication stress response, at least partly through PCNA-Ub-dependent lesion bypass, to ensure genome stability. Deregulation of SDE2 levels, either by knockdown or by defective proteolysis, disrupts this genome maintenance pathway.</p

A PIP box in the SDE2-UBL is required for the cleavage of SDE2.

<p><b>(A)</b> A schematic showing a PIP box in the SDE2-UBL. PIP boxes from different species are aligned, and consensus elements are marked with asterisks. <b>(B)</b> 293T cell lysates expressing GFP-SDE2 GA or GA + PIP (F47A & F48A) mutants were incubated with GST- or GST-PCNA-bound glutathione beads and analyzed by Western blotting. <b>(C)</b> HeLa cells expressing GFP-tagged SDE2 wild-type or PIP mutant were analyzed by Western blotting. <b>(D)</b> GFP-SDE2 variants transfected into U2OS cells were fixed and visualized by fluorescence microscopy. <b>(E)</b> SDE2-Flag proteins <i>in vitro</i> transcribed and translated (IVTT) from reticulocyte lysates were analyzed by Western blotting. Where indicated, ubiquitin vinyl sulfone (Ub-VS) was added during expression. <b>(F)</b> Inhibition of cleavage of IVTT SDE2-Flag GA and PIP mutants. ΔSAP: aa395-450 deletion.</p