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

Rageul, Julie; Park, Jennifer J.; Zeng, Ping Ping; Lee, Euna; Yang, Jinho; Hwang, Sunyoung; Lo, Natalie; Weinheimer, Alexandra S.; Schärer, Orlando D.; Yeo, Jung-Eun; Kim, Hyungjin

doi:10.1038/s41467-020-19162-5

Cited by 38 publications

(37 citation statements)

References 61 publications

(73 reference statements)

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“…Importantly, a TIM-EdU PLA assay to visualize TIM at EdU-labeled ongoing replication forks demonstrated that unlike the wild-type, the ΔSAP mutant fails to complement the percentage of cells with positive PLA foci to the level of control following siRNA knockdown and dox induction, indicating that SDE2 SAP is necessary for the localization of TIM at replication forks, similar to the phenotype of the ΔSDE2 domain mutant as we previously showed ( Fig. 7 , C and D ) (19). Accordingly, DNA combing analysis, which allows for monitoring dynamics of single DNA replication tracks, revealed that cells re-expressing the ΔSAP mutant exhibit a significant shortening of replication tracks in comparison to wild-type cells, indicating that SDE2 SAP is required for the function of TIM to support efficient fork progression ( Fig.…”

Section: Resultssupporting

confidence: 82%

Extended DNA binding interface beyond the canonical SAP domain contributes to SDE2 function at DNA replication forks

Weinheimer

Paung

Rageul

et al. 2022

Preprint

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Elevated DNA replication stress causes instability of the DNA replication fork and DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator SDE2 binds to TIMELESS (TIM) of the fork protection complex (FPC) and enhances its stability, thereby supporting replisome activity at DNA replication forks. Here, we structurally and functionally characterize a new conserved DNA binding motif related to SAP (SAF-A/B, Acinus, PIAS) in human SDE2 and establish its preference for single-stranded DNA (ssDNA). The nuclear magnetic resonance solution structure of SDE2SAP reveals a helix-extended loop-helix core aligned parallel to each other, consistent with known canonical SAP folds. Notably, its DNA interaction extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to SAP and conserved in the pre-mRNA splicing factor SF3A3. Mutation in the SAP domain with extended C-terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Together, our study establishes SDE2SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via FPC regulation and highlights the structural diversity of the DNA-protein interactions achieved by a specialized DNA binding motif.

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Section: Resultssupporting

confidence: 82%

Extended DNA binding interface beyond the canonical SAP domain contributes to SDE2 function at DNA replication forks

Weinheimer

Paung

Rageul

et al. 2022

Preprint

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“…The high resolution of our cryo‐EM map has enabled us to build an atomic model for the core human replisome that provides a wealth of information showing how five key replisome proteins are organised by CMG, the complex protein:protein interactions that underpin this organisation and how replication fork DNA is coordinated for template unwinding. The stable positioning of TIMELESS‐TIPIN at the front of the replisome enables it to grip dsDNA before unwinding and this is likely to be important for TIMELESS‐TIPIN‐dependent replication fork stabilisation and fork protection (Leman et al , 2010 ; Rageul et al , 2020 ), perhaps by stabilising the entire replisome on chromatin when its progression is perturbed. The C‐terminal ˜400 a.a. of TIMELESS contains a DBD and PBD.…”

Section: Discussionmentioning

confidence: 99%

Structure of a human replisome shows the organisation and interactions of a DNA replication machine

et al. 2021

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The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45‐MCM‐GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading‐strand polymerase Pol ε, together with TIMELESS‐TIPIN, CLASPIN and AND‐1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Å cryo‐EM structure of a human replisome comprising CMG, Pol ε, TIMELESS‐TIPIN, CLASPIN and AND‐1 bound to replication fork DNA. The structure permits a detailed understanding of how AND‐1, TIMELESS‐TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS‐TIPIN with replication fork DNA suggests a mechanism for strand separation.

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“…Timeless (Fig. 4 C) plays an important role in DNA replication via maintenance of replication fork and genome stability [ 31 , 32 ]. Protein DBF4 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Proteomic analysis of bone marrow-derived mesenchymal stem cell extracellular vesicles from healthy donors: implications for proliferation, angiogenesis, Wnt signaling, and the basement membrane

et al. 2021

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Background Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown therapeutic potential in various in vitro and in vivo studies in cutaneous wound healing. Furthermore, there are ubiquitous studies highlighting the pro-regenerative effects of BM-MSC extracellular vesicles (BM-MSC EVs). The similarities and differences in BM-MSC EV cargo among potential healthy donors are not well understood. Variation in EV protein cargo is important to understand, as it may be useful in identifying potential therapeutic applications in clinical trials. We hypothesized that the donors would share both important similarities and differences in cargo relating to cell proliferation, angiogenesis, Wnt signaling, and basement membrane formation—processes shown to be critical for effective cutaneous wound healing. Methods We harvested BM-MSC EVs from four healthy human donors who underwent strict screening for whole bone marrow donation and further Good Manufacturing Practices-grade cell culture expansion for candidate usage in clinical trials. BM-MSC EV protein cargo was determined via mass spectrometry and Proteome Discoverer software. Corresponding proteomic networks were analyzed via the UniProt Consortium and STRING consortium databases. Results More than 3000 proteins were identified in each of the donors, sharing > 600 proteins among all donors. Despite inter-donor variation in protein identities, there were striking similarities in numbers of proteins per biological functional category. In terms of biologic function, the proteins were most associated with transport of ions and proteins, transcription, and the cell cycle, relating to cell proliferation. The donors shared essential cargo relating to angiogenesis, Wnt signaling, and basement membrane formation—essential processes in modulating cutaneous wound repair. Conclusions Healthy donors of BM-MSC EVs contain important similarities and differences among protein cargo that may play important roles in their pro-regenerative functions. Further studies are needed to correlate proteomic signatures to functional outcomes in cutaneous repair.

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

Cited by 38 publications

References 61 publications

Extended DNA binding interface beyond the canonical SAP domain contributes to SDE2 function at DNA replication forks

Extended DNA binding interface beyond the canonical SAP domain contributes to SDE2 function at DNA replication forks

Structure of a human replisome shows the organisation and interactions of a DNA replication machine

Proteomic analysis of bone marrow-derived mesenchymal stem cell extracellular vesicles from healthy donors: implications for proliferation, angiogenesis, Wnt signaling, and the basement membrane

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