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.

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 TIM stability and its localization to replication forks, thereby aiding 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. form a four-way junction by the action of the RAD51 recombinase and several SWI/SNF family translocases such as SMARCAL1, ZRANB3, and HLTF 14,15 . This unique transaction protects stressed forks from degradation and acts as an intermediate for the repair and restart of stalled forks [16][17][18] . The tumor suppressor proteins BRCA1/2 and Fanconi anemia (FA) protein FANCD2 are required for the protection of reversed forks by stabilizing the RAD51 filaments and preventing the regressed arm from nucleolytic degradation 19 . Many regulatory proteins fine-tune the steps of processing or protecting reversed forks, and deregulated fork remodeling has been associated with fork collapse, genome instability, and sensitivity or resistance to chemotherapy 20,21 .The fork protection complex (FPC), composed of TIMELESS (TIM) and TIPIN (Swi1 and Swi3 in S. pombe, and Tof1 and Csm3 in S. cerevisiae), and the ancillary proteins AND-1 and CLASPIN (CLSPN) stabilizes the replisome to ensure unperturbed fork progression [22][23][24] . The FPC acts as a scaffold to link the movements of the CMG helicase and polymerase, preventing the uncoupling of their activities and ensuring efficient replisome progression 25,26 . Additionally, the FPC promotes the ATR-CHK1 checkpoint signaling under replication stress by stimulating the association of TIM-TIPIN and CLSPN to RPA-ssDNA at stalled forks, thereby facilitating CLSPN-mediated CHK1 phosphorylation by ATR 27,28 . TIM and TIPIN form an obligate heterodimer and have no known enzymatic function, suggesting that they play a structural role in supporting replisome integrity 29 . Loss of the FPC leads to defects in DNA replication and genome instability, indicating that maintaining structural integrity of the replisome is critical for preserving fork stability [30][31][32] . TIM is upregulated in a variety of cancers, implying that enhanced FPC activity may alleviate replication stress arising in tumors through oncogene activation 33 . However, the regu...