Current status and prospect of the DNA double-strand break repair pathway in colorectal cancer development and treatment.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair.

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

Functional genetic variants of GEN1 predict overall survival of Chinese epithelial ovarian cancer patients.

BACKGROUND: Inherited variations in DNA double-strand break (DSB) repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. Despite its significance, survival-associated genetic variants within the DSB pathway remain underexplored. METHODS: In the present study, we performed a two-phase analysis of 19,290 single-nucleotide polymorphisms (SNPs) in 199 genes in the DSB repair pathway from a genome-wide association study (GWAS) dataset and explored their associations with overall survival (OS) in 1039 Han Chinese epithelial ovarian carcinoma (EOC) patients. After utilizing multivariate Cox regression analysis with bayesian false-discovery probability for multiple test correction, significant genetic variations were identified and subsequently underwent functional prediction and validation. RESULTS: We discovered a significant association between poor overall survival and the functional variant GEN1 rs56070363 C > T (CT + TT vs. TT, adjusted hazard ratio (HR) = 2.50, P < 0.001). And the impact of GEN1 rs56070363 C > T on survival was attributed to its reduced binding affinity to hsa-miR-1287-5p and the resultant upregulation of GEN1 mRNA expression. Overexpression of GEN1 aggregated EOC cell proliferation, invasion and migration presumably by influencing the expression of immune inhibitory factors, thereby elevating the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and then constructing an immunosuppressive tumor microenvironment. CONCLUSIONS: In conclusion, GEN1 rs56070363 variant could serve as a potential predictive biomarker and chemotherapeutic target for improving the survival of EOC patients.

Exploring factors influencing choice of DNA double-strand break repair pathways.

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9's remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies.

(Single-stranded DNA) gaps in understanding BRCAness.

The tumour-suppressive roles of BRCA1 and 2 have been attributed to three seemingly distinct functions - homologous recombination, replication fork protection, and single-stranded (ss)DNA gap suppression - and their relative importance is under debate. In this review, we examine the origin and resolution of ssDNA gaps and discuss the recent advances in understanding the role of BRCA1/2 in gap suppression. There are ample data showing that gap accumulation in BRCA1/2-deficient cells is linked to genomic instability and chemosensitivity. However, it remains unclear whether there is a causative role and the function of BRCA1/2 in gap suppression cannot unambiguously be dissected from their other functions. We therefore conclude that the three functions of BRCA1 and 2 are closely intertwined and not mutually exclusive.

Macromutations Yielding Karyotype Alterations (and the Process(es) behind Them) Are the Favored Route of Carcinogenesis and Speciation.

It is argued that carcinogenesis and speciation are evolutionary events which are based on changes in the 'karyotypic code' through a phase of 'genome instability', followed by a bottleneck of selection for the viability and adaptability of the initial cells. Genomic (i.e., chromosomal) instability is caused by (massive) DNA breakage and the subsequent mis-repair of DNA double-strand breaks (DSBs) resulting in various chromosome rearrangements. Potential tumor cells are selected for rapid somatic proliferation. Cells eventually yielding a novel species need not only to be viable and proliferation proficient, but also to have a balanced genome which, after passing meiosis as another bottleneck and fusing with an identical gamete, can result in a well-adapted organism. Such new organisms should be genetically or geographically isolated from the ancestral population and possess or develop an at least partial sexual barrier.

Exploring DNA repair deficient CHO cell response to low dose rate radiation.

INTRODUCTION: DNA double-strand breaks (DSBs) induced by ionizing radiation pose a significant threat to genome integrity, necessitating robust repair mechanisms. This study explores the responses of repair-deficient cells to low dose rate (LDR) radiation. Non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways play pivotal roles in maintaining genomic stability. The hypothesis posits distinct cellular outcomes under LDR exposure compared to acute radiation, impacting DNA repair mechanisms and cell survival. MATERIALS AND METHODS: Chinese hamster ovary (CHO) cells, featuring deficiencies in NHEJ, HR, Fanconi Anemia, and PARP pathways, were systematically studied. Clonogenic assays for acute and LDR gamma-ray exposures, cell growth inhibition analyses, and γ-H2AX foci assays were conducted, encompassing varied dose rates to comprehensively assess cellular responses. RESULTS: NHEJ mutants exhibited an unexpected inverse dose rate effect, challenging conventional expectations. HR mutants displayed unique radiosensitivity patterns, aligning with responses to major DNA-damaging agents. LDR exposure induced cell cycle alterations, growth delays, and giant cell formation, revealing context-dependent sensitivities. γ-H2AX foci assays indicated DSB accumulation during LDR exposure. DISCUSSION: These findings challenge established paradigms, emphasizing the intricate interplay between repair pathways and dose rates. The study offers comprehensive insights into repair-deficient cell responses, urging a reevaluation of conventional dose-response models and providing potential avenues for targeted therapeutic strategies in diverse radiation scenarios.

EXO1 protects BRCA1-deficient cells against toxic DNA lesions.

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.

Templated insertions-DNA repair gets acrobatic.

Deletions associated with the repair of DNA double-strand breaks is a source of genetic alternation and a recognized source of disease-causing mutagenesis. Theta-mediated end joining is a DNA repair mechanism, which guarantees deletions by its employment of microhomology (MH) alignment to facilitate end joining. A lesser-characterized templated insertion ability of this pathway, on the other hand, is associated with both deletion and insertion. This mechanism is characterized by at least one round of polymerase θ-mediated synthesis, which does not result in successful repair, followed by a subsequent round of polymerase engagement and synthesis that does lead to repair. Here we focus on the mechanisms by which polymerase θ introduces these insertions-direct, inverse, and a new class which we have termed strand switching. We observe this new class of templated insertions at multiple loci and across multiple species, often at a comparable frequency to those previously characterized. Templated insertion mutations are often enriched in cancer genomes and repeat expansion disorders. This repair mechanism thus contributes to disease-associated mutagenesis, and may plausibly even promote disease. Characterization of the types of polymerase θ-dependent insertions can provide new insight into these diseases and clinical promise for treatment.

Evidence for increased DNA damage repair in the postmortem brain of the high stress-response group of schizophrenia.

Background: Schizophrenia (SZ) is a disorder diagnosed by specific symptoms and duration and is highly heterogeneous, clinically and pathologically. Although there are an increasing number of studies on the association between genetic and environmental factors in the development of SZ, the actual distribution of the population with different levels of influence of these factors has not yet been fully elucidated. In this study, we focused on stress as an environmental factor and stratified SZ based on the expression levels of stress-responsive molecules in the postmortem prefrontal cortex. Methods: We selected the following stress-responsive molecules: interleukin (IL) -1ß, IL-6, IL-10, tumor necrosis factor-α, interferon-γ, glucocorticoid receptor, brain-derived neurotrophic factor, synaptophysin, S100 calcium-binding protein B, superoxide dismutase, postsynaptic density protein 95, synuclein, apolipoprotein A1 (ApoA1), ApoA2, and solute carrier family 6 member 4. We performed RNA sequencing in the prefrontal gray matter of 25 SZ cases and 21 healthy controls and conducted a hierarchical cluster analysis of SZ based on the gene expression levels of stress-responsive molecules, which yielded two clusters. After assessing the validity of the clusters, they were designated as the high stress-response SZ group and the low stress-response SZ group, respectively. Ingenuity Pathway Analysis of differentially expressed genes (DEGs) between clusters was performed, and Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was conducted on four cases each in the high and low stress-response SZ groups to validate DNA damage. Results: We found higher prevalence of family history of SZ in the low stress-response SZ group (0/3 vs. 5/4, p = 0.04). Pathway analysis of DEGs between clusters showed the highest enrichment for DNA double-strand break repair. TUNEL staining showed a trend toward a lower percentage of TUNEL-positive cells in the high stress-response SZ group. Conclusion: Our results suggest that there are subgroups of SZ with different degrees of stress impact. Furthermore, the pathophysiology of these subgroups may be associated with DNA damage repair. These results provide new insights into the interactions and heterogeneity between genetic and environmental factors.

Holding it together: DNA end synapsis during non-homologous end joining.

DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the majority of these breaks in vertebrates by directly ligating DNA ends back together. Upon formation of a DSB, a multiprotein complex is assembled on DNA ends which tethers them together within a synaptic complex. Synapsis is a critical step of the NHEJ pathway as loss of synapsis can result in mispairing of DNA ends and chromosome translocations. As DNA ends are commonly incompatible for ligation, the NHEJ machinery must also process ends to enable rejoining. This review describes how recent progress in single-molecule approaches and cryo-EM have advanced our molecular understanding of DNA end synapsis during NHEJ and how synapsis is coordinated with end processing to determine the fidelity of repair.

The Impact of NAD Bioavailability on DNA Double-Strand Break Repair Capacity in Human Dermal Fibroblasts after Ionizing Radiation.

Nicotinamide adenine dinucleotide (NAD) serves as a substrate for protein deacetylases sirtuins and poly(ADP-ribose) polymerases, which are involved in the regulation of DNA double-strand break (DSB) repair molecular machinery by various mechanisms. However, the impact of NAD bioavailability on DSB repair remains poorly characterized. Herein, using immunocytochemical analysis of γH2AX, a marker for DSB, we investigated the effect of the pharmacological modulation of NAD levels on DSB repair capacity in human dermal fibroblasts exposed to moderate doses of ionizing radiation (IR). We demonstrated that NAD boosting with nicotinamide riboside did not affect the efficiency of DSB elimination after the exposure of cells to IR at 1 Gy. Moreover, even after irradiation at 5 Gy, we did not observe any decrease in intracellular NAD content. We also showed that, when the NAD pool was almost completely depleted by inhibition of its biosynthesis from nicotinamide, cells were still able to eliminate IR-induced DSB, though the activation of ATM kinase, its colocalization with γH2AX and DSB repair capacity were reduced in comparison to cells with normal NAD levels. Our results suggest that NAD-dependent processes, such as protein deacetylation and ADP-ribosylation, are important but not indispensable for DSB repair induced by moderate doses of IR.

The DHX9 helicase interacts with human DNA polymerase δ4 and stimulates its activity in D-loop extension synthesis.

The extension of the invading strand within a displacement loop (D-loop) is a key step in homology directed repair (HDR) of doubled stranded DNA breaks. The primary goal of these studies was to test the hypotheses that 1) D-loop extension by human DNA polymerase δ4 (Pol δ4) is facilitated by DHX9, a 3' to 5' motor helicase, which acts to unwind the leading edge of the D-loop, and 2) the recruitment of DHX9 is mediated by direct protein-protein interactions between DHX9 and Pol δ4 and/or PCNA. DNA synthesis by Pol δ4 was analyzed in a reconstitution assay by the extension of a 93mer oligonucleotide inserted into a plasmid to form a D-loop. Product formation by Pol δ4 was monitored by incorporation of [α-32P]dNTPs into the 93mer primer followed by denaturing gel electrophoresis. The results showed that DHX9 strongly stimulated Pol δ4 mediated D-loop extension. Direct interactions of DHX9 with PCNA, the p125 and the p12 subunits of Pol δ4 were demonstrated by pull-down assays with purified proteins. These data support the hypothesis that DHX9 helicase is recruited by Pol δ4/PCNA to facilitate D-loop synthesis in HDR, and is a participant in cellular HDR. The involvement of DHX9 in HDR represents an important addition to its multiple cellular roles. Such helicase-polymerase interactions may represent an important aspect of the mechanisms involved in D-loop primer extension synthesis in HDR.

APTX acts in DNA double-strand break repair in a manner distinct from XRCC4.

Aprataxin (APTX), the product of the causative gene for hereditary neurogenerative syndromes Ataxia-oculomotor apraxia 1 and early onset ataxia with oculomotor apraxia and hypoalbuminemia, has an enzymatic activity of removing adenosine monophosphate from DNA 5'-end, which arises from abortive ligation by DNA ligases. It is also reported that APTX physically binds to XRCC1 and XRCC4, suggesting its involvement in DNA single-strand break repair (SSBR) and DNA double-strand break repair (DSBR) via non-homologous end joining pathway. Although the involvement of APTX in SSBR in association with XRCC1 has been established, the significance of APTX in DSBR and its interaction with XRCC4 have remained unclear. Here, we generated APTX knock-out (APTX-/-) cell from human osteosarcoma U2OS through CRISPR/Cas9-mediated genome editing system. APTX-/- cells exhibited increased sensitivity toward ionizing radiation (IR) and Camptothecin in association with retarded DSBR, as shown by increased number of retained γH2AX foci. However, the number of retained 53BP1 foci in APTX-/- cell was not discernibly different from wild-type cells, in stark contrast to XRCC4-depleted cells. The recruitment of GFP-tagged APTX (GFP-APTX) to the DNA damage sites was examined by laser micro-irradiation and live-cell imaging analysis using confocal microscope. The accumulation of GFP-APTX on the laser track was attenuated by siRNA-mediated depletion of XRCC1, but not XRCC4. Moreover, the deprivation of APTX and XRCC4 displayed additive inhibitory effects on DSBR after IR exposure and end joining of GFP reporter. These findings collectively suggest that APTX acts in DSBR in a manner distinct from XRCC4.

SLFN5-mediated chromatin dynamics sculpt higher-order DNA repair topology.

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.

ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen.

ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis.

Chromosomal Rearrangements and Chromothripsis: The Alternative End Generation Model.

Chromothripsis defines a genetic phenomenon where up to hundreds of clustered chromosomal rearrangements can arise in a single catastrophic event. The phenomenon is associated with cancer and congenital diseases. Most current models on the origin of chromothripsis suggest that prior to chromatin reshuffling numerous DNA double-strand breaks (DSBs) have to exist, i.e., chromosomal shattering precedes rearrangements. However, the preference of a DNA end to rearrange in a proximal accessible region led us to propose chromothripsis as the reaction product of successive chromatin rearrangements. We previously coined this process Alternative End Generation (AEG), where a single DSB with a repair-blocking end initiates a domino effect of rearrangements. Accordingly, chromothripsis is the end product of this domino reaction taking place in a single catastrophic event.

Geriatric Frailty Is Associated With Oxidative Stress, Accumulation, and Defective Repair of DNA Double-Strand Breaks Independently of Age and Comorbidities.

Defects in the DNA damage response and repair (DDR/R) network accumulate during the aging process. Physical frailty, a state of reduced physiological function and decreased resilience to biological stressors, is also exacerbated by aging, but its link with DDR/R aberrations beyond the effect of age and comorbidities is unclear. Fifty-three community-dwelling older adults, aged 65-102 years, who underwent frailty classification according to the Rockwood Clinical Frailty Scale (CFS), and 51 healthy adults younger than 45 years were examined in parallel. The following DDR/R parameters were determined in their peripheral blood mononuclear cells (PBMCs): (a) oxidative stress and abasic (apurinic/apyrimidinic; AP) sites, (b) endogenous DNA damage (alkaline comet assay olive tail moment [OTM] indicative of DNA single-strand breaks [SSBs] and double-strand breaks [DSBs] and γH2AX levels by immunofluorescence [DSBs only]), (c) capacity of the 2 main DNA repair mechanisms (DSB repair and nucleotide excision repair). Older individual-derived PBMCs displayed reduced-to-oxidized glutathione ratios indicative of increased levels of oxidative stress and increased AP sites, as well as increased accumulation of endogenous DNA damage (OTM and γH2AX) and defective DSB-repair capacity, compared with younger controls. These DDR/R aberrations were more pronounced in frail versus nonfrail older adults. Notably, oxidative stress, AP sites, DSBs, and DSB-repair capacity were associated with individual CFS levels after adjusting for chronological age, sex, Charlson Comorbidity Index, and polypharmacy. Geriatric frailty is independently associated with increased DNA damage formation and reduced DSB-R capacity, supporting further research into these measures as potential frailty biomarkers.

A moving target for drug discovery: Structure activity relationship and many genome (de)stabilizing functions of the RAD52 protein.

BRCA-ness phenotype, a signature of many breast and ovarian cancers, manifests as deficiency in homologous recombination, and as defects in protection and repair of damaged DNA replication forks. A dependence of such cancers on DNA repair factors less important for survival of BRCA-proficient cells, offers opportunities for development of novel chemotherapeutic interventions. The first drugs targeting BRCA-deficient cancers, poly-ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of advanced, chemotherapy resistant cancers in patients with BRCA1/2 germline mutations. Nine additional proteins that can be targeted to selectively kill BRCA-deficient cancer cells have been identified. Among them, a DNA repair protein RAD52 is an especially attractive target due to general tolerance of the RAD52 loss of function, and protective role of an inactivating mutation. Yet, the effective pharmacological inhibitors of RAD52 have not been forthcoming. In this review, we discuss advances in the state of our knowledge of the RAD52 structure, activities and cellular functions, with a specific focus on the features that make RAD52 an attractive, but difficult drug target.

Epstein-Barr virus protein BKRF4 restricts nucleosome assembly to suppress host antiviral responses.

Inhibition of host DNA damage response (DDR) is a common mechanism used by viruses to manipulate host cellular machinery and orchestrate viral life cycles. Epstein-Barr virus tegument protein BKRF4 associates with cellular chromatin to suppress host DDR signaling, but the underlying mechanism remains elusive. Here, we identify a BKRF4 histone binding domain (residues 15-102, termed BKRF4-HBD) that can accumulate at the DNA damage sites to disrupt 53BP1 foci formation. The high-resolution structure of the BKRF4-HBD in complex with a human H2A-H2B dimer shows that BKRF4-HBD interacts with the H2A-H2B dimer via the N-terminal region (NTR), the DWP motif (residues 80-86 containing D81, W84, P86), and the C-terminal region (CTR). The "triple-anchor" binding mode confers BKRF4-HBD the ability to associate with the partially unfolded nucleosomes, promoting the nucleosome disassembly. Importantly, disrupting the BKRF4-H2A-H2B interaction impairs the binding between BKRF4-HBD and nucleosome in vitro and inhibits the recruitment of BKRF4-HBD to DNA breaks in vivo. Together, our study reveals the structural basis of BKRF4 bindings to the partially unfolded nucleosome and elucidates an unconventional mechanism of host DDR signal attenuation.