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.

DNA replication is a tightly controlled process that ensures the faithful duplication of the genome. However, DNA damage arising from both endogenous and exogenous assaults gives rise to DNA replication stress associated with replication fork slowing or stalling. Therefore, protecting the stressed fork while prompting its recovery to complete DNA replication is critical for safeguarding genomic integrity and cell survival. Specifically, the plasticity of the replication fork in engaging distinct DNA damage tolerance mechanisms, including fork reversal, repriming, and translesion DNA synthesis, enables cells to overcome a variety of replication obstacles. Furthermore, stretches of single-stranded DNA generated upon fork stalling trigger the activation of the ATR kinase, which coordinates the cellular responses to replication stress by stabilizing the replication fork, promoting DNA repair, and controlling cell cycle and replication origin firing. Deregulation of the ATR checkpoint and aberrant levels of chronic replication stress is a common characteristic of cancer and a point of vulnerability being exploited in cancer therapy. Here, we discuss the various adaptive responses of a replication fork to replication stress and the roles of ATR signaling that bring fork stabilization mechanisms together. We also review how this knowledge is being harnessed for the development of checkpoint inhibitors to trigger the replication catastrophe of cancer cells.

Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover.

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 SDE2^SAP 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 SDE2^SAP 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.

Oncogene-induced senescence (OIS) is a potent barrier that limits tumorigenesis. While persistent DNA replication stress accompanied by cellular hyperproliferation is considered an underlying basis of OIS, the molecular mechanisms through which oncogenes elicit genome instability remain unclear. Here, we identify a distinct cellular process in which DNA replication fork stalling is linked to senescence in response to oncogenic signaling. We demonstrate that TIMELESS (TIM), an essential regulatory component of the fork protection complex that supports the integrity of the replisome, is downregulated in non-transformed human cell lines that undergo OIS by HRAS^G12V expression. Loss of TIM is sufficient to induce senescence, while ectopic expression of TIM delays the induction of OIS via enhancing protection of stalled forks. Furthermore, TIM levels are restored in cells capable of bypassing OIS, indicating that TIM regulation modulates the onset of OIS. Mechanistically, HRAS^G12V expression induces cellular poly(ADP-ribosyl)ation mediated by PARP1, which prompts poly(ADP-ribose)/PAR-dependent TIM degradation. Together, our study uncovers RAS-mediated proteolytic signaling as a key determinant of OIS centered at stalled DNA replication forks. We propose the existence of an oncogenic signaling cascade that actively suppresses replisome activity, contributing to the DNA replication stress that drives OIS.