Double-strand Break Repair Research Articles

HP1 Promotes the Centromeric Localization of ATRX and Protects Cohesion by Interfering Wapl Activity in Mitosis.

α thalassemia/mental retardation syndrome X-linked (ATRX) serves as a part of the sucrose nonfermenting 2 (SNF2) chromatin-remodeling complex. In interphase, ATRX localizes to pericentromeric heterochromatin, contributing to DNA double-strand break repair, DNA replication, and telomere maintenance. During mitosis, most ATRX proteins are removed from chromosomal arms, leaving a pool near the centromere region in mammalian cells, which is critical for accurate chromosome congression and sister chromatid cohesion protection. However, the function and localization mechanisms of ATRX at mitotic centromeres remain largely unresolved. The clustered regularly interspaced short palindromic repeats with CRISPR-associated protein 9 (CRISPR-Cas9) system and overexpression approaches were employed alongside immunofluorescence to investigate the mechanism of ATRX localization at the centromere. To study the binding mechanism between ATRX and heterochromatin protein 1 (HP1), both full-length and truncated mutants of hemagglutinin (HA)-ATRX were generated for co-immunoprecipitation and glutathione S-transferase (GST)-pull assays. Wild-type ATRX and HP1 binding-deficient mutants were created to investigate the role of ATRX binding to HP1 during mitosis, with the Z-Leu-Leu-Leu-al (MG132) maintenance assay, cohesion function assay, and kinetochore distance measurement. Our research demonstrated that HP1α, HP1β, and HP1γ facilitate the positioning of ATRX within the mitotic centromere area through their interaction with the first two [P/L]-X-V-X-[M/L/V] (PxVxL)motifs at the N-terminus of ATRX. ATRX deficiency causes aberrant mitosis and decreased centromeric cohesion. Furthermore, reducing Wapl activity can bypass the need for ATRX to protect centromeric cohesion. These results provide insights into the mechanism of ATRX's centromeric localization and its critical function in preserving centromeric cohesion by reducing Wapl activity in human cells.

A dual role of Cohesin in DNA DSB repair

Cells undergo tens of thousands of DNA-damaging events each day. Defects in repairing double-stranded breaks (DSBs) can lead to genomic instability, contributing to cancer, genetic disorders, immunological diseases, and developmental defects. Cohesin, a multi-subunit protein complex, plays a crucial role in both chromosome organization and DNA repair by creating architectural loops through chromatin extrusion. However, the mechanisms by which cohesin regulates these distinct processes are not fully understood. In this study, we identify two separate roles for cohesin in DNA repair within mammalian cells. First, cohesin serves as an intrinsic architectural factor that normally prevents interactions between damaged chromatin. Second, cohesin has an architecture-independent role triggered by ATM phosphorylation of SMC1, which enhances the efficiency of repair. Our findings suggest that these two functions work together to reduce the occurrence of translocations and deletions associated with non-homologous end joining, thereby maintaining genomic stability.

Defective homologous recombination and genomic instability predict increased responsiveness to carbon ion radiotherapy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is notably resistant to conventional chemotherapy and radiation treatment. However, clinical trials indicate that carbon ion radiotherapy (CIRT) with concurrent gemcitabine is effective for unresectable locally advanced PDAC. This study aimed to identify patient characteristics predictive of CIRT response. We utilized a panel of human PDAC cell lines with diverse genetic profiles to determine their sensitivity to CIRT compared to γ-rays, assessing relative biological effectiveness (RBE) at 10% survival, which ranged from 1.96 to 3.04. Increased radiosensitivity was linked to impaired DNA double-strand break (DSB) repair, particularly in cell lines with deficiencies in the homologous recombination (HR) repair pathway and/or elevated genomic instability from replication stress. Furthermore, pretreatment with the HR inhibitor B02 significantly enhanced CIRT sensitivity in a radioresistant PDAC cell line when irradiated in the spread-out Bragg peak but not at the entry position of the beam. These findings suggest that PDAC tumors with HR pathway mutations or high replication stress are more likely to benefit from CIRT while minimizing normal tissue toxicity.

A mobile genetic element-derived primase-polymerase harbors multiple activities implicated in DNA replication and repair.

Primase-polymerases (PrimPols) play divergent functions from DNA replication to DNA repair in all three life domains. In archaea and bacteria, numerous and diverse PPs are encoded by mobile genetic elements (MGEs) and act as the replicases for their MGEs. However, their varying activities and functions are not fully understood. In this study, we characterized a group of PrimPols that are genetically associated with prokaryotic argonaute proteins (pAgos). The pAgo-associated PrimPol (AgaPP) is likely derived from a MGE. AgaPP has polymerase and primase activities and physically interacts with a helicase encoded by its downstream gene, suggesting that they constitute a functional replication module. Further, AgaPP performs translesion DNA synthesis, terminal transfer and microhomology-mediated end joining (MMEJ), showing striking similarity to human DNA repair polymerase θ. AgaPP can promote the MMEJ repair of Cas9-induced double-stranded DNA breaks and increase cell survival post DNA damage in Escherichia coli. In addition, the MMEJ activity of AgaPP can be repurposed to assist DNA assembly in vitro. Together, the findings reveal dual role of AgaPP in both DNA replication and repair.

GDBr: genomic signature interpretation tool for DNA double-strand break repair mechanisms.

Large genetic variants can be generated via homologous recombination (HR), such as polymerase theta-mediated end joining (TMEJ) or single-strand annealing (SSA). Given that these HR-based mechanisms leave specific genomic signatures, we developed GDBr, a genomic signature interpretation tool for DNA double-strand break repair mechanisms using high-quality genome assemblies. We applied GDBr to a draft human pangenome reference. We found that 78.1% of non-repetitive insertions and deletions and 11.0% of non-repetitive complex substitutions contained specific signatures. Of these, we interpreted that 98.7% and 1.3% of the insertions and deletions were generated via TMEJ and SSA, respectively, and all complex substitutions via TMEJ. Since population-level pangenome datasets are being dramatically accumulated, GDBr can provide mechanistic insights into how variants are formed. GDBr is available on GitHub at https://github.com/Chemical118/GDBr.

Kinetic analysis of strand invasion during C. elegans meiosis reveals similar rates of sister- and homolog-directed repair.

Meiotic chromosome segregation requires reciprocal exchanges between the parental chromosomes (homologs). Exchanges are formed via tightly-regulated repair of double-strand DNA breaks (DSBs). However, since repair intermediates are mostly quantified in fixed images, our understanding of the mechanisms that control the progression of repair remains limited. Here, we study meiotic repair kinetics in Caenorhabditis elegans by extinguishing new DSBs and following the disappearance of a crucial intermediate - strand invasion mediated by the conserved RecA-family recombinase RAD-51. We find that RAD-51 foci have a half-life of 42-132 minutes for both endogenous and exogenous DSBs. Surprisingly, we find that repair templated by the sister chromatid is not slower than repair templated by the homolog. This suggests that differential kinetics are unlikely to underlie 'homolog bias': the preferential use of the homolog as a repair template. We also use our kinetic information to revisit the total number of DSBs per nucleus - the 'substrate' for the formation of exchanges - and find an average of 40 DSBs in wild-type meiosis and >50 DSBs when homolog pairing is perturbed. Our work opens the door for analysis of the interplay between meiotic repair kinetics and the fidelity of genome inheritance.

REV7: a small but mighty regulator of genome maintenance and cancer development.

REV7, also known as MAD2B, MAD2L2, and FANCV, is a HORMA-domain family protein crucial to multiple genome stability pathways. REV7's canonical role is as a member of polymerase ζ, a specialized translesion synthesis polymerase essential for DNA damage tolerance. REV7 also ensures accurate cell cycle progression and prevents premature mitotic progression by sequestering an anaphase-promoting complex/cyclosome activator. Additionally, REV7 supports genome integrity by directing double-strand break repair pathway choice as part of the recently characterized mammalian shieldin complex. Given that genome instability is a hallmark of cancer, it is unsurprising that REV7, with its numerous genome maintenance roles, is implicated in multiple malignancies, including ovarian cancer, glioma, breast cancer, malignant melanoma, and small-cell lung cancer. Moreover, high REV7 expression is associated with poor prognoses and treatment resistance in these and other cancers. Promisingly, early studies indicate that REV7 suppression enhances sensitivity to chemotherapeutics, including cisplatin. This review aims to provide a comprehensive overview of REV7's myriad roles in genome maintenance and other functions as well as offer an updated summary of its connections to cancer and treatment resistance.

Argonaute 2 regulates nuclear DNA damage, repair, and phenotypes in Arabidopsis under genotoxic stress.

Argonaute (AGO) proteins are involved in gene expression and genome integrity during biotic and abiotic stress responses. AGO2 mediates double-strand break (DSB) repair in DNA damage response (DDR) induced by genotoxic stress. However, beyond DSB repair, the involvement of AGO proteins in DDR remains unknown. To investigate the potential roles and functions of AGO2 in DDR, we exposed three different ago2 mutants, each harboring a T-DNA insertion in the promoter, the N-terminal domain of exon 2, or the P-element-induced wimpy testis (PIWI) domain of exon 3, to genotoxic stress, and examined their DDR phenotypes. DDR phenotypes, such as root cell death and growth inhibition following γ-irradiation and zeocin treatment, were significantly suppressed by defects in the promoter or N-terminal domain of AGO2 but not by defects in the PIWI domain, which is responsible for RNA silencing. The weak DDR phenotypes were rescued by AGO2 overexpression and were attributed to reduced nuclear DNA damage despite impaired DNA repair, including DSB repair, as shown in comet and γH2AX assays. These results suggest that AGO2 regulates overall nuclear DNA damage and DDR phenotypes beyond DSB repair through the N-terminal domain rather than the PIWI domain. The potential role of AGO2 in the DDR implies that DNA repair may not be the primary factor for determining susceptibility to genotoxic stress.

Gestational exposure to arsenic reduces female offspring fertility by impairing the repair of DNA double-strand breaks and synapsis formation in oocytes

Arsenic is a pollutant that can cross the placenta; however, research on the effects of arsenic exposure during pregnancy on the fertility of female offspring is limited. To address this gap, we developed a mouse model to investigate the relationship between arsenic exposure during pregnancy and fertility in female offspring. Our fertility assessment revealed that gestational exposure to 1mg/kg arsenic or higher (10mg/kg) resulted in reduction in litter size, ovarian volume, and multistage-follicle number in female offspring. By assessing the in vitro developmental capacity of oocytes and zygotes, we confirmed that the reduced fertility was due not to impaired oocyte quality but rather to a reduction in oocyte quantity. Arsenic exposure impedes synapsis formation in MPI and compromises homologous recombination-mediated repair of double-strand breaks, resulting in fewer crossovers. This disruption activates the pachytene-checkpoint, hindering the progression of the MPI and resulting in the elimination of defective oocytes through p-Chk2 activation. Our study reveals for the first time the detrimental effects of arsenic exposure during pregnancy on the fertility of female offspring, underscoring the urgent need to prevent such exposure to safeguard reproductive health.

The interplay between chromatin remodeling and DNA double-strand break repair: Implications for cancer biology and therapeutics.

Proper chromatin remodeling is crucial for many cellular physiological processes, including the repair of DNA double-strand break (DSB). While the mechanism of DSB repair is well understood, the connection between chromatin remodeling and DSB repair remains incompletely elucidated. In this review, we aim to highlight recent studies demonstrating the close relationship between chromatin remodeling and DSB repair. We summarize the impact of DSB repair on chromatin, including nucleosome arrangement, chromatin organization, and dynamics, and conversely, the role of chromatin architecture in regulating DSB repair. Additionally, we also summarize the contribution of chromatin remodeling complexes to cancer biology through DNA repair and discuss their potential as therapeutic targets for cancer.

Single-Molecule Visualization of BLM-DNA2-Mediated DNA End Resection Using DNA Curtains.

Homologous recombination (HR) is the principal pathway undertaken by a cell for the error-free repair of DNA double-strand breaks that are frequently encountered by the cell. HR can be initiated at the sites of DNA double-strand breaks by generating long stretches of single-stranded 3' DNA overhang through a process called DNA end resection. In one DNA end resection pathway, this is achieved via the concerted effort of specialized machinery involving the RecQ family helicase BLM, the helicase/endonuclease DNA2, and a single-strand DNA binding protein complex RPA. BLM unwinds the DNA at the sites of DNA damage. The DNA unwound by BLM is cleaved in a 5' to 3' direction by DNA2 leaving behind a long single-stranded 3' DNA tail which is rapidly coated with RPA. This long single-stranded DNA provides the loading platform for recombinase proteins to form nucleoprotein filaments which initiate the homology search to undergo HR-based repair. Most of the insights into these processes have been gained using ensemble biochemical methods. However, these approaches have proven to be challenging for dissecting complex molecular mechanisms, especially because of the dynamic nature of these processes. Experiments involving single-molecule techniques have enabled us to counter these issues by allowing us to gather information on intermediates that are heterogeneous and transient in nature. We have developed a single-molecule technique called DNA curtains where we can study hundreds of protein-nucleic acid interaction events in real time. Here, we describe a method to prepare a single-tethered double-stranded DNA curtain to visualize, in combination with total internal reflection microscopy (TIRFM), the BLM- and DNA2-mediated DNA end resection in real time.

Homologous recombination promotes non-immunogenic mitotic cell death upon DNA damage

Double-strand breaks (DSBs) can initiate mitotic catastrophe, a complex oncosuppressive phenomenon characterized by cell death during or after cell division. Here we unveil how cell cycle-regulated DSB repair guides disparate cell death outcomes through single-cell analysis of extended live imaging. Following DSB induction in S or G2, passage of unresolved homologous recombination intermediates into mitosis promotes non-immunogenic intrinsic apoptosis in the immediate attempt at cell division. Conversely, non-homologous end joining, microhomology-mediated end joining and single-strand annealing cooperate to enable damaged G1 cells to complete the first cell cycle with an aberrant cell division at the cost of delayed extrinsic lethality and interferon production. Targeting non-homologous end joining, microhomology-mediated end joining or single-strand annealing promotes mitotic death, while suppressing mitotic death enhances interferon production. Together the data indicate that a temporal repair hierarchy, coupled with cumulative DSB load, serves as a reliable predictor of mitotic catastrophe outcomes following genome damage. In this pathway, homologous recombination suppresses interferon production by promoting mitotic lethality.

Discovery of new inhibitors of nuclease MRE11.

MRE11 nuclease is a central player in signaling and processing DNA damage, and in resolving stalled replication forks. Here, we describe the identification and characterization of new MRE11 inhibitors MU147 and MU1409. Both compounds inhibit MRE11 nuclease more specifically and effectively than the relatively weak state-of-the-art inhibitor mirin. They also abrogate double-strand break repair mechanisms that rely on MRE11 nuclease activity, without impairing ATM activation. Inhibition of MRE11 also impairs nascent strand degradation of stalled replication forks and selectively affects BRCA2-deficient cells. Herein, we illustrate that our newly discovered compounds MU147 and MU1409 can be used as chemical probes to further explore the biological role of MRE11 and support the potential clinical relevance of pharmacological inhibition of this nuclease.

How to shift the equilibrium of dna break repair in favor of homology recombination

With the practical implementation of the CRISPR/Cas technology for targeted genome editing, it has become possible to carry out genetic engineering manipulations with eukaryotic genomes with high efficiency. One of the key stages of this technology is the targeted induction of site-specific DNA cleavages (breaks). The cell repairs these breaks via one of two pathways: nonhomologous end joining or homologous recombination. The choice of DNA repair pathway is determined by the architecture of the sites at the DNA break area formed as a result of terminal resection and depends on the phases of the cell cycle. Nonhomologous end joining is the main pathway for repair of double-stranded DNA breaks in mammalian cells. It involves a nonspecific ligation reaction, the accuracy of which depends on the structure of the ends of the break, and can result in various insertions or deletions in the target region of the genome. Integration of the desired sequence into the genome occurs along the path of homologous recombination, the implementation of which requires a matrix with homology regions on both sides of the double-strand break. The introduction of a genetic construct into a given location in the genome is an important, but currently complex and labor-intensive task. At the same time, for fundamental studies of gene function and the creation of animal models of human diseases, the choice of the repair pathway can be of fundamental importance. This review is an attempt to combine and structure all known information on approaches to increasing the efficiency of DNA repair involving homologous recombination. The article lists the most effective strategies to shift the balance towards homologous repair, such as the use of inhibitors of the non-homologous end joining mechanism, regulation of key factors of homologous recombination, control of the cell cycle, chromatin status, construction of templates for homologous recombination.

Huntingtin interactome reveals huntingtin role in regulation of double strand break DNA damage response (DSB/DDR), chromatin remodeling and RNA processing pathways.

Huntington's Disease (HD), a progressive neurodegenerative disorder with no disease-modifying therapies, is caused by a CAG repeat expansion in the HD gene encoding polyglutamine-expanded huntingtin (HTT) protein. Mechanisms of HD cellular pathogenesis and cellular functions of the normal and mutant HTT proteins are still not completely understood. HTT protein has numerous interaction partners, and it likely provides a scaffold for assembly of multiprotein complexes many of which may be altered in HD. Previous studies have implicated DNA damage response in HD pathogenesis. Gene transcription and RNA processing has also emerged as molecular mechanisms associated with HD. Here we used multiple approaches to identify HTT interactors in the context of DNA damage stress. Our results indicate that HTT interacts with many proteins involved in the regulation of interconnected DNA repair/remodeling and RNA processing pathways. We present evidence for a role for HTT in double strand break repair mechanism. We demonstrate HTT functional interaction with a major DNA damage response kinase DNA-PKcs and association of both proteins with nuclear speckles. We show that S1181 phosphorylation of HTT is regulated by DSB, and can be carried out (at least in vitro) by DNA-PK. Furthermore, we show HTT interactions with RNA binding proteins associated with nuclear speckles, including two proteins encoded by genes at HD modifier loci, TCERG1 and MED15, and with chromatin remodeling complex BAF. These interactions of HTT may position it as an important scaffolding intermediary providing integrated regulation of gene expression and RNA processing in the context of DNA repair mechanisms.

Statistical Analysis of Gene Variants for Homologous Recombination Pathways of DNA Repair leading to Cancer Susceptibility

Background: RAD51C, a critical member of the RAD51 paralog family, is essential for homologous recombination (HR)-mediated DNA repair, a pathway crucial for maintaining genomic stability. Mutations in RAD51C have been linked to cancer susceptibility, particularly in breast and ovarian cancers, where impaired DNA repair mechanisms contribute to genomic instability and tumor progression. Despite its clinical significance, the functional impact of specific RAD51C variants remains poorly understood, necessitating a comprehensive investigation into their biological implications. Methods: This study classified RAD51C gene variants into damaging and tolerant categories using computational prediction tools, including SIFT, PolyPhen, CADD, MetaLR, and Mutation Assessor. Variants were prioritized based on consensus scores and classified as high-confidence damaging variants. Correlation and agreement among tools were analyzed to refine predictions. Principal Component Analysis (PCA) and clustering methods were employed to group variants based on prediction patterns. Protein-protein interaction (PPI) networks and pathway enrichment analyses were conducted to contextualize damaging variants within broader biological systems, with a focus on their roles in HR, DNA repair, and cellular processes. Results: A total of 2526 variants were analyzed, with damaging variants showing consistent patterns across tools. Consensus scores highlighted 302 high-confidence damaging variants, which were associated with disrupted biological processes, including double-strand break repair via homologous recombination, telomere maintenance, and regulation of cell cycle checkpoints. PPI analysis revealed an interconnected network with 11 nodes and 54 edges, with a clustering coefficient of 0.982, indicating tightly coordinated interactions among DNA repair proteins. Pathway enrichment analyses identified significant associations with homologous recombination (FDR = 2.55E-17) and the Fanconi anemia pathway (FDR = 2.96E-06). Conclusion: This study provides a comprehensive framework for assessing the functional impacts of RAD51C variants by integrating computational predictions with biological analyses. The findings underscore the importance of RAD51C in HR and DNA repair pathways, offering insights into its role in genomic stability and cancer progression. These results can inform the prioritization of variants for experimental validation and guide therapeutic strategies targeting DNA repair deficiencies.

Post-Integrational DNA Repair of HIV-1 Is Associated with the Activation of DNA-PK and ATM Cellular Protein Kinases and Phosphorylation of Their Targets

Integration of the DNA copy of the HIV-1 genome into the cellular genome results in series of damages, the repair of which is critical for successful viral replication. We have previously demonstrated that the ATM and DNA-PK kinases, normally responsible for repairing double-strand breaks in the cellular DNA, are required to initiate HIV-1 post-integration repair, even though integration does not result in double-strand DNA breaks. In this study, we analyzed changes in the phosphorylation status of ATM (pSer1981), DNA-PK (pSer2056) and their related kinase ATR (pSer428), as well as their targets: Chk1 (pSer345), Chk2 (pThr68), H2AX (pSer139) and p53 (pSer15) during HIV-1 post-integration repair. We have shown that ATM and DNA-PK, but not ATR, undergo autophosphorylation during postintegration DNA repair and phosphorylate their target proteins Chk2 and H2AX. These data indicate common signaling mechanisms between double-strand DNA break repair and postintegration repair of HIV-1.

Beyond Nucleotide Excision Repair: The Importance of XPF in Base Excision Repair and Its Impact on Cancer, Inflammation, and Aging.

DNA repair involves various intricate pathways that work together to maintain genome integrity. XPF (ERCC4) is a structural endonuclease that forms a heterodimer with ERCC1 that is critical in both single-strand break repair (SSBR) and double-strand break repair (DSBR). Although the mechanistic function of ERCC1/XPF has been established in nucleotide excision repair (NER), its role in long-patch base excision repair (BER) has recently been discovered through the 5'-Gap pathway. This study briefly explores the roles of XPF in different pathways to emphasize the importance of XPF in DNA repair. XPF deficiency manifests in various diseases, including cancer, neurodegeneration, and aging-related disorders; it is also associated with conditions such as Xeroderma pigmentosum and fertility issues. By examining the molecular mechanisms and pathological consequences linked to XPF dysfunction, this study aims to elucidate the crucial role of XPF in genomic stability as a repair protein in BER and provide perspectives regarding its potential as a therapeutic target in related diseases.

BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL).

Increased expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) often correlates with tumor aggressiveness and drug resistance in cancer. We have recently reported that BCAT1 was overexpressed in a subgroup of T-cell acute lymphoblastic (T-ALL) samples, especially those with NOTCH1 activating mutations. Interestingly, BCAT1-depleted cells showed pronounced sensitivity to DNA-damaging agents such as etoposide; however, how BCAT1 regulates this sensitivity remains uncertain. Here, we provide further clues on its chemo-sensitizing effect. Indeed, BCAT1 protein regulates the non-homologous end joining (c-NHEJ) DNA repair pathway by physically associating with the KU70/KU80 heterodimer. BCAT1 inhibition during active repair of DNA double-strand breaks (DSBs) led to increased KU70/KU80 acetylation and impaired c-NHEJ repair, a dramatic increase in DSBs, and ultimately cell death. Our results suggest that, in T-ALL, BCAT1 possesses non-metabolic functions that confer a drug resistance mechanism and that targeting BCAT1 activity presents a novel strategy to improve chemotherapy response in T-ALL patients.

RRM1 promotes homologous recombination and radio/chemo-sensitivity via enhancing USP11 and E2F1-mediated RAD51AP1 transcription

Ribonucleotide reductase M1 (RRM1), the catalytic subunit of ribonucleotide reductase, plays a pivotal role in converting ribonucleotides (NTP) into deoxyribonucleotides (dNTP), essential for DNA replication and repair. Elevated RRM1 expression is associated with various human cancers, correlating with poorer prognosis and reduced overall survival rates. Our previous study found that RRM1 will enter the nucleus to promote DNA damage repair. However, the underlying mechanism remains elusive. Here, we unveil a novel role of RRM1 in promoting homologous recombination (HR) by upregulating the expression of RAD51AP1, a critical HR factor, in an E2F1-dependent manner. We demonstrate that RRM1 interacts with USP11 in the cytoplasm, and the recruitment of RRM1 to LaminB1 induced by ionizing radiation (IR) facilitates the binding of USP11 to the nuclear pore complex (NPC), promoting USP11 entry into the nucleus. Upon nuclear translocation, USP11 binds to E2F1 and inhibits the ubiquitin-mediated degradation of E2F1, thereby enhancing the transcriptional expression of RAD51AP1. Moreover, a specific RRM1 mutant lacking amino acids 731–793, crucial for its interaction with USP11 and recruitment to LaminB1, exhibits a dominant-negative effect on RAD51AP1 expression and HR. Truncations of RRM1 fail to inhibit the ubiquitin-mediated degradation of E2F1 and cannot promote the E2F1-mediated transactivation of RAD51AP1. Lastly, the full length of RRM1, not truncations, enhances tumor cells’ sensitivity to IR, underscoring its importance in radiotherapy resistance. Collectively, our results suggest a novel function of RRM1 in promoting HR-mediated DSB repair through positive regulation of RAD51AP1 transcription by direct interaction with USP11 and promoting subsequent USP11-mediated deubiquitination of E2F1. Our findings elucidate a previously unknown mechanism whereby RRM1 promotes HR-mediated DNA repair, presenting a potential therapeutic target for cancer treatment.