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1.
J Biol Chem ; 298(8): 102268, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35850305

RESUMEN

Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA-binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for ssDNA. Our NMR solution structure of the SDE2SAP domain reveals a helix-extended loop-helix core with the helices aligned parallel to each other, consistent with known canonical SAP folds. Notably, we have shown that the DNA interaction of this SAP domain extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to the SAP motif and conserved in the pre-mRNA splicing factor SF3A3. Furthermore, we found that mutation in the SAP domain and extended C terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Taken together, our results establish SDE2SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via fork protection complex regulation and highlight the structural diversity of the DNA-protein interactions achieved by a specialized DNA-binding motif.


Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Dominios Proteicos
2.
Nat Commun ; 11(1): 5495, 2020 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-33127907

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Estructuras Cromosómicas/metabolismo , Daño del ADN , Reparación del ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica/fisiología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Dominios Proteicos
3.
DNA Repair (Amst) ; 81: 102657, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31324531

RESUMEN

Faithful duplication of the genome is critical for the survival of an organism and prevention of malignant transformation. Accurate replication of a large amount of genetic information in a timely manner is one of the most challenging cellular processes and is often perturbed by intrinsic and extrinsic barriers to DNA replication fork progression, a phenomenon referred to as DNA replication stress. Elevated DNA replication stress is a primary source of genomic instability and one of the key hallmarks of cancer. Therefore, targeting DNA replication stress is an emerging concept for cancer therapy. The replication machinery associated with PCNA and other regulatory factors coordinates the synthesis and repair of DNA strands at the replication fork. The dynamic interaction of replication protein complexes with DNA is essential for sensing and responding to various signaling events relevant to DNA replication and damage. Thus, the disruption of the spatiotemporal regulation of protein homeostasis at the replication fork impairs genome integrity, which often involves the deregulation of ubiquitin-mediated proteolytic signaling. Notably, emerging evidence has highlighted the role of the AAA+ATPase VCP/p97 in extracting ubiquitinated protein substrates from the chromatin and facilitating the turnover of genome surveillance factors during DNA replication and repair. Here, we review recent advances in our understanding of chromatin-associated degradation pathways at the replication fork and the implication of these findings for cancer therapy.


Asunto(s)
Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Replicación del ADN , Animales , Eucariontes/genética , Eucariontes/metabolismo , Inestabilidad Genómica , Humanos , Proteolisis , Ubiquitina/metabolismo
4.
Nucleic Acids Res ; 47(8): 3996-4010, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-30698750

RESUMEN

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-SDE2Ct, the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2Ct 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/p97UFD1-NPL4 segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2Ct degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2Ct, 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.


Asunto(s)
Proteínas de Unión al ADN/genética , ADN/genética , Genoma , Proteína de Replicación A/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cromatina/efectos de la radiación , ADN/metabolismo , Replicación del ADN/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/efectos de la radiación , Osteoblastos , Fosforilación/efectos de la radiación , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteolisis/efectos de la radiación , Proteína de Replicación A/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Rayos Ultravioleta , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismo
5.
Biochemistry ; 56(35): 4637-4645, 2017 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-28759203

RESUMEN

The suppressor of T cell signaling (Sts) proteins, Sts-1 and Sts-2, are homologous phosphatases that negatively regulate signaling pathways downstream of the T cell receptor. Functional inactivation of Sts-1 and Sts-2 in a murine model leads to resistance to systemic infection by the opportunistic pathogen, Candida albicans. This suggests that modulation of the host immune response by inhibiting Sts function may be a viable strategy for treating these deadly fungal pathogen infections. To better understand the molecular determinants of function and structure, we characterized the structure and steady-state kinetics of the histidine phosphatase domains of human Sts-1 (Sts-1HP) and Sts-2 (Sts-2HP). We determined the X-ray crystal structures of unliganded Sts-1HP and Sts-1HP in complex with sulfate to 2.5 and 1.9 Å, respectively, and the structure of Sts-2HP with sulfate to 2.4 Å. The steady-state kinetic analysis shows, as expected, that Sts-1HP has a phosphatase activity significantly higher than that of Sts-2HP and that the human and mouse proteins behave similarly. In addition, comparison of the phosphatase activity of full-length Sts-1 protein to Sts-1HP reveals similar kinetics, indicating that Sts-1HP is a functional surrogate for the native protein. We also tested known phosphatase inhibitors and determined that the SHP-1 inhibitor, PHPS1, is a potent inhibitor of Sts-1 (Ki = 1.05 ± 0.15 µM). Finally, we demonstrated that human Sts-1 has robust phosphatase activity against the substrate, Zap-70, in a cell-based assay. Collectively, these data suggest that the human Sts proteins are druggable targets and provide a structural basis for future drug development efforts.


Asunto(s)
Proteínas Portadoras/química , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Fosfatasas/química , Dominio Catalítico , Clonación Molecular , Humanos , Proteínas de la Membrana , Modelos Moleculares , Monoéster Fosfórico Hidrolasas/química , Conformación Proteica , Dominios Proteicos
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