DOT1L-mediated RAP80 methylation promotes BRCA1 recruitment to elicit DNA repair.

Breast Cancer Type 1 Susceptibility Protein (BRCA1) is a tumor-suppressor protein that regulates various cellular pathways, including those that are essential for preserving genome stability. One essential mechanism involves a BRCA1-A complex that is recruited to double-strand breaks (DSBs) by RAP80 before initiating DNA damage repair (DDR). How RAP80 itself is recruited to DNA damage sites, however, is unclear. Here, we demonstrate an intrinsic correlation between a methyltransferase DOT1L-mediated RAP80 methylation and BRCA1-A complex chromatin recruitment that occurs during cancer cell radiotherapy resistance. Mechanistically, DOT1L is quickly recruited onto chromatin and methylates RAP80 at multiple lysines in response to DNA damage. Methylated RAP80 is then indispensable for binding to ubiquitinated H2A and subsequently triggering BRCA1-A complex recruitment onto DSBs. Importantly, DOT1L-catalyzed RAP80 methylation and recruitment of BRCA1 have clinical relevance, as inhibition of DOT1L or RAP80 methylation seems to enhance the radiosensitivity of cancer cells both in vivo and in vitro. These data reveal a crucial role for DOT1L in DDR through initiating recruitment of RAP80 and BRCA1 onto chromatin and underscore a therapeutic strategy based on targeting DOT1L to overcome tumor radiotherapy resistance.

Crosstalk between Lys63- and Lys11-polyubiquitin signaling at DNA damage sites is driven by Cezanne.

The establishment of polyubiquitin conjugates with distinct linkages play important roles in the DNA damage response. Much remains unknown about the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Here we reveal that Cezanne (also known as Otud7B) deubiquitinating enzyme promotes the recruitment of Rap80/BRCA1-A complex by binding to Lys63-polyubiquitin and targeting Lys11-polyubiquitin. Using a ubiquitin binding domain protein array screen, we identify that the UBA domains of Cezanne and Cezanne2 (also known as Otud7A) selectively bind to Lys63-linked polyubiquitin. Increased Lys11-linkage ubiquitination due to lack of Cezanne DUB activity compromises the recruitment of Rap80/BRCA1-A. Cezanne2 interacts with Cezanne, facilitating Cezanne in the recruitment of Rap80/BRCA1-A, Rad18, and 53BP1, in cellular resistance to ionizing radiation and DNA repair. Our work presents a model that Cezanne serves as a "reader" of the Lys63-linkage polyubiquitin at DNA damage sites and an "eraser" of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites.

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.

RAP80 and BRCA1 PARsylation protect chromosome integrity by preventing retention of BRCA1-B/C complexes in DNA repair foci.

BRCA1 promotes error-free, homologous recombination-mediated repair (HRR) of DNA double-stranded breaks (DSBs). When excessive and uncontrolled, BRCA1 HRR activity promotes illegitimate recombination and genome disorder. We and others have observed that the BRCA1-associated protein RAP80 recruits BRCA1 to postdamage nuclear foci, and these chromatin structures then restrict the amplitude of BRCA1-driven HRR. What remains unclear is how this process is regulated. Here we report that both BRCA1 poly-ADP ribosylation (PARsylation) and the presence of BRCA1-bound RAP80 are critical for the normal interaction of BRCA1 with some of its partners (e.g., CtIP and BACH1) that are also known components of the aforementioned focal structures. Surprisingly, the simultaneous loss of RAP80 and failure therein of BRCA1 PARsylation results in the dysregulated accumulation in these foci of BRCA1 complexes. This in turn is associated with the intracellular development of a state of hyper-recombination and gross chromosomal disorder. Thus, physiological RAP80-BRCA1 complex formation and BRCA1 PARsylation contribute to the kinetics by which BRCA1 HRR-sustaining complexes normally concentrate in nuclear foci. These events likely contribute to aneuploidy suppression.

RAP80 binds p32 to preserve the functional integrity of mitochondria.

RAP80, a member of the BRCA1-A complex, is a well-known crucial regulator of cell cycle checkpoint and DNA damage repair in the nucleus. However, it is still unclear whether Rap80 localizes to another region outside the nucleus and plays different roles with its partners. Here, we found mitochondrial p32 as a novel binding partner of RAP80 by using yeast two-hybrid screening. RAP80 directly binds the internal region of p32 through its arginine rich C-terminal domain. Based on the interaction, we showed that a subset of RAP80 localizes to mitochondria where p32 exists. Loss of function study revealed that RAP80 deficiency reduces the protein level of p32 and p32 dependent mitochondrial translating proteins such as Rieske and COX1. As a result, mitochondrial membrane potential and oxygen consumption are reduced in RAP80 knockdown cells, indicating mitochondrial dysfunction. Our study identifies a novel interaction between RAP80 and p32, which is important for preserving intact mitochondrial function.

A BRCA1-interacting lncRNA regulates homologous recombination.

Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.

RAP80 regulates epithelial-mesenchymal transition related with metastasis and malignancy of cancer.

Epithelial-mesenchymal transition (EMT) has been closely related with invasive and metastatic properties of cancer. Recently, the convergence of DNA damage response and EMT in cancer development has received a great amount of scientific attention. Here, we showed that EMT is induced by the downregulation of RAP80, a well-known regulator for DNA damage response. The knockdown of RAP80 leads to EMT-like morphological changes and the increase of tumor sphere formation in non-adhesive culture. Mechanistically, RAP80 controls a reciprocal regulatory axis of ZEB1 (for EMT activation) and miR200c (for EMT inhibition). The downregulation of RAP80 increases ZEB1 protein and decreases miR200c expression to activate EMT signaling in the form of drastic inhibitions of E-cadherin, p16 and p21 expression. Using in vivo metastasis analysis, RAP80 knockdown cells are shown to dramatically metastasize into the lung and generate more malignant phenotype compared to controls. Interestingly, the expression level of RAP80 was positively correlated with the survival rate in lung adenocarcinoma and breast cancer patients. These findings indicate that RAP80 is a critical gatekeeper in impeding EMT-induced metastasis and malignant phenotypes of cancer as well as preserving DNA integrity.

DNA repair pathways to regulate response to chemoradiotherapy in patients with locally advanced head and neck cancer.

Platinum-based chemoradiotherapy (CRT) is a preferred standard of care for locally advanced head and neck cancer (HNC). However, survival benefit is small, with substantial toxicity and biomarkers of CRT resistance that could guide treatment selection and spare morbidity. Increased DNA repair in solid tumors may contribute to cancer cells' ability to survive in genotoxic stress environments afforded by therapy. We assessed mRNA expression levels of DNA repair-related genes BRCA1, RAP80, 53 binding protein 1 (53BP1), mediator of DNA damage checkpoint 1 (MDC1), and RNF8. We correlated our findings with response and overall survival in 72 head and neck patients treated with weekly carboplatin AUC 2 and radiotherapy. Complete response (CR) to CRT was 50 % in patients with low levels of 53BP1 compared to 6.3 % in patients with high levels (p = 0.0059). Of high BRCA1 mRNA expressors, 41.2 % had CR compared to 29.4 % of low expressors (p = 0.72). For a small group of patients with low 53BP1 and either high BRCA1 or RAP80, CRs were 66.7 and 71.4 %, respectively. A trend for better overall survival (OS) was found for patients with low 53BP1 (15 vs 8 m; p = 0.056). Our findings highlight the potential usefulness of 53BP1 mRNA as a predictive biomarker of response and overall survival in HNC patients treated with chemoradiotherapy. Those with high 53BP1 expression could derive only a meager benefit from treatment. Analysis of BRCA1 and RAP80 could further reinforce the predictive value of 53BP1. Although this was a retrospective study with small sample size, it could inform larger translational studies in HNC.

Factors forming the BRCA1-A complex orchestrate BRCA1 recruitment to the sites of DNA damage.

Sustaining genomic integrity is essential for preventing onset of cancers. Therefore, human cells evolve to have refined biological pathways to defend genetic materials from various genomic insults. DNA damage response and DNA repair pathways essential for genome maintenance are accomplished by cooperative executions of multiple factors including breast cancer type 1 susceptibility protein (BRCA1). BRCA1 is initially identified as an altered gene in the hereditary breast cancer patients. Since then, tremendous efforts to understand the functions of BRAC1 reveal that BRCA1 is found in distinct complexes, including BRCA1-A, BRCA1-B, BRCA1-C, and the BRCA1/PALB2/BRCA2 complex, and plays diverse roles in a context-dependent manner. Among the complexes, BRCA1-A is critical for BRCA1 recruitment to the sites of DNA damage. Factors comprising the BRCA1-A include RAP80, CCDC98/Abraxas, BRCC36, BRCC45, BARD1, BRCA1, and MERIT40, a RAP80-associated factor. In this review, we summarize recent findings of the factors that form the BRCA1-A complex.

Two biomarker-directed randomized trials in European and Chinese patients with nonsmall-cell lung cancer: the BRCA1-RAP80 Expression Customization (BREC) studies.

BACKGROUND: In a Spanish Lung Cancer Group (SLCG) phase II trial, the combination of BRCA1 and receptor-associated protein 80 (RAP80) expression was significantly associated with outcome in Caucasian patients with nonsmall-cell lung cancer (NSCLC). The SLCG therefore undertook an industry-independent collaborative randomized phase III trial comparing nonselected cisplatin-based chemotherapy with therapy customized according to BRCA1/RAP80 expression. An analogous randomized phase II trial was carried out in China under the auspices of the SLCG to evaluate the effect of BRCA1/RAP80 expression in Asian patients. PATIENTS AND METHODS: Eligibility criteria included stage IIIB-IV NSCLC and sufficient tumor specimen for molecular analysis. Randomization to the control or experimental arm was 1 : 1 in the SLCG trial and 1 : 3 in the Chinese trial. In both trials, patients in the control arm received docetaxel/cisplatin; in the experimental arm, patients with low RAP80 expression received gemcitabine/cisplatin, those with intermediate/high RAP80 expression and low/intermediate BRCA1 expression received docetaxel/cisplatin, and those with intermediate/high RAP80 expression and high BRCA1 expression received docetaxel alone. The primary end point was progression-free survival (PFS). RESULTS: Two hundred and seventy-nine patients in the SLCG trial and 124 in the Chinese trial were assessable for PFS. PFS in the control and experimental arms in the SLCG trial was 5.49 and 4.38 months, respectively [log rank P = 0.07; hazard ratio (HR) 1.28; P = 0.03]. In the Chinese trial, PFS was 4.74 and 3.78 months, respectively (log rank P = 0.82; HR 0.95; P = 0.82). CONCLUSION: Accrual was prematurely closed on the SLCG trial due to the absence of clinical benefit in the experimental over the control arm. However, the BREC studies provide proof of concept that an international, nonindustry, biomarker-directed trial is feasible. Thanks to the groundwork laid by these studies, we expect that ongoing further research on alternative biomarkers to elucidate DNA repair mechanisms will help define novel therapeutic approaches. TRIAL REGISTRATION: NCT00617656/GECP-BREC and ChiCTR-TRC-12001860/BREC-CHINA.

Role of MERIT40 in stabilization of BRCA1 complex: a protein-protein interaction study.

MERIT40 is a novel associate of the BRCA1-complex, thus play an essential role in DNA damage repair mechanism. It is the least implicit protein and its structural and functional aspects of regulating the stability of BRCA1-MERIT40 complex remain equivocal. Analysis of protein-protein interactions between BRCA1 and its cellular binding partners like ABRAXAS, RAP80 and MERIT40 would help to understand the role of protein complex integrity in DNA repair mechanism. The recombinant proteins were purified and their structural aspects were elucidated by spectroscopic methods. Interaction analysis was carried out to determine binding partners of MERIT40. MERIT40 showed interaction with bridging molecule, called ABRAXAS, thus generate a scaffold among various members which further stabilizes the entire complex. It acts as an adapter molecule by interacting with BRCA1-BRCT in non-phosphorylation dependent manner. The feature enlighten on structural and interaction profile of BRCA1-complex member to elucidate their role in complex stability and DNA repair process.

RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair.

Single-stranded DNA (ssDNA) arising as an intermediate of cellular processes on DNA is a potential vulnerability of the genome unless it is appropriately protected. Recent evidence suggests that R-loops, consisting of ssDNA and DNA-RNA hybrids, can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. However, how the vulnerability of ssDNA in R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops, chromosome translocations, and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

In vitro assembly complex formation of TRAIP CC and RAP 80 zinc finger motif revealed by our study.

BACKGROUND: Tumor necrosis factor interacting protein (TRAIP/TRIP) is an important cell-signaling molecule that prevents the TNF-induced-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation via direct interaction with TRAF 2 protein. TRAIP is a crucial downstream signaling molecule, implicated in several signaling pathways. Due to these multifunctional effects, TRAIP is more related to cellular mitosis, chromosome segregation, and DNA damage response. Tumor necrosis factor interacting protein is a downstream signaling molecule that contains a RING domain with E3 ubiquitin ligase activity at the N terminal side followed by coiled-coil and C terminal leucine zipper domain. Human TRAIP is constituted of 469 amino acids with 76% sequence similarity with the mouse TRAIP protein. Although, the main inhibitory function of TRAIP has been known for decades, however, in vitro interaction of TRAIPCC domain with RAP80 Zinc finger motif has not been reported yet. Besides, RAP80, the binding partner of TRAIPCC protein has been implicated in DNA damage response. RESULTS: Our in vitro study shows that the TRAIP CC (64-166) associates with the RAP80 zinc finger of corresponding amino acid 490-584. However, TRAIP CCLZ (66-260) and TRAIP RINGCC (1 = 157) failed to interact with the RAP80 zinc finger of corresponding amino acid 490-584. The current study reinforces TRAIP CC (64-166) and RAP80 zinc finger of corresponding amino acid 490-584 associates to form a complex. Moreover, SDS PAGE arbitrated the homogeneity of RAP80 Zinc finger and TRAIP CC of corresponding amino acid 490-584 and 64-166, respectively. CONCLUSION: In vitro, a specific interaction was observed between the TRAIP CC (64-166) and the RAP80 zinc finger of the corresponding amino acid 490-584 and a specific binding area of the RAP80 zinc finger motif were investigated. The TRAIPCC region is required for the complex to bind to the RAP80-Zn finger motif. This strategy may be necessary for the RAP80 zinc finger activity to the TRAIP CC protein.

BRCA1-A and BRISC: Multifunctional Molecular Machines for Ubiquitin Signaling.

The K63-linkage specific deubiquitinase BRCC36 forms the core of two multi-subunit deubiquitination complexes: BRCA1-A and BRISC. BRCA1-A is recruited to DNA repair foci, edits ubiquitin signals on chromatin, and sequesters BRCA1 away from the site of damage, suppressing homologous recombination by limiting resection. BRISC forms a complex with metabolic enzyme SHMT2 and regulates the immune response, mitosis, and hematopoiesis. Almost two decades of research have revealed how BRCA1-A and BRISC use the same core of subunits to perform very distinct biological tasks.

RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells.

BACKGROUND: RAP80 is a member of BRCA1-A complex, which plays an important role in regulating the cell cycle checkpoint and DNA damage repair in the nucleus. METHOD: We investigated RAP80 expression in breast cancer and its paired normal breast tissues to further analyze its role in the biological behavior of breast cancer cells. RESULTS: RAP80 expression in breast cancer (62.3%, 101/162) was significantly lower than that in adjacent normal breast tissues (P<0.05). RAP80 expression was related to tumor size, lymph node metastasis, TNM stage, and molecular subtype (P<0.05). RAP80 mRNA expression was significantly lower in triple-negative breast cancer than other types. The mRNA and protein of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231, so we picked MCF-7 to be transfected with RAP80 siRNA. The survival rate of both cells decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83 µg/mL, and 1.69 µg/mL in wild-type MCF-7 according to MTT. RAP80 siRNA transfection upregulated the apoptosis and downregulated invasive or migrating ability of MCF-7. RAP80 siRNA also upregulated the protein expression of Caspase-3, cleaved Caspase-3, Apaf-1, Cytochrome C, Bax, and Fas, and downregulated the protein expression of Bcl-2. CONCLUSION: RAP80 expression was related to ER or PR activity. Inhibition of RAP80 expression can induce apoptosis in breast cancer cells and improve chemosensitivity to cisplatin. Tumor cells can activate protective responses to inhibit cell cycle progression, which may be related to RAP80, and repair cisplatin-induced DNA damage. RAP80 is related to BRCA1's effect, which can be used as an interesting target for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy.

Association between EGFR mutation and expression of BRCA1 and RAP80 in non-small cell lung cancer.

Association between the epithelial growth factor receptor (EGFR) mutation and the expression of breast cancer 1 (BRCA1) and receptor-associated protein 80 (RAP80) in non-small cell lung cancer (NSCLC) was studied. From September 2014 to September 2016, 51 patients with NSCLC who were hospitalized in Department of Thoracic Surgery in The Affiliated Jiangyin Hospital of Southeast University Medical College and underwent biopsy or operation were selected. The pathological changes of lung tissue were detected by hematoxylin and eosin histopathological staining. The fluorescent expression of BRCA1 and RAP80 protein in the two groups was determined by immunofluorescence staining. Reverse transcriptase polymerase chain reaction method and western blot analysis were used to detect BRCA1 and RAP80 mRNA and protein expression. Then the EGFR gene mutation was detected and analyzed. The results show that non-small cell lung cancer has an association with smoking. Compared with the control, the lung tissue structure of the NSCLC group was damaged. The protein fluorescence expression of BRCA1 and RAP80 in non-small cell lung cancer group was significantly increased. The expression of BRCA1 and RAP80 mRNA and protein in NSCLC group was significantly increased. The difference in expression of BRCA1 and RAP80 in the control and the non-small cell lung cancer group was statistically significant (P<0.05). EGFR gene mutations detected 14 of the 51 patients with genetic mutations. Non-small cell lung cancer and smoking have certain relevance, and BRCA1 and RAP80 expression in the development and progression of NSCLC has a close relationship. EGFR mutation in non-small cell lung cancer significantly related to the mutation of EGFR and BRCA1 and RAP80 gene expression plays an important role in the diagnosis and treatment of NSCLC.

RAP80, ubiquitin and SUMO in the DNA damage response.

A decade has passed since the first reported connection between RAP80 and BRCA1 in DNA double-strand break repair. Despite the initial identification of RAP80 as a factor localizing BRCA1 to DNA double-strand breaks and potentially promoting homologous recombination, there is increasing evidence that RAP80 instead suppresses homologous recombination to fine-tune the balance of competing DNA repair processes during the S/G2 phase of the cell cycle. RAP80 opposes homologous recombination by inhibiting DNA end-resection and sequestering BRCA1 into the BRCA1-A complex. Ubiquitin and SUMO modifications of chromatin at DNA double-strand breaks recruit RAP80, which contains distinct sequence motifs that recognize ubiquitin and SUMO. Here, we review RAP80's role in repressing homologous recombination at DNA double-strand breaks and how this role is facilitated by its ability to bind ubiquitin and SUMO modifications.

Choreographing the Double Strand Break Response: Ubiquitin and SUMO Control of Nuclear Architecture.

The cellular response to DNA double strand breaks (DSBs) is a multifaceted signaling program that centers on post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. In this review we discuss how ubiquitin and SUMO orchestrate the recognition of DSBs and explore how this influences chromatin organization. We discuss functional outcomes of this response including transcriptional silencing and how pre-existing chromatin states may control the DSB response and the maintenance of genomic stability.

Structural and functional characterization of the MERIT40 to understand its role in DNA repair.

MERIT40 (MEdiator of RAP80 Interaction and Targeting 40) is a novel associate of the BRCA1-complex and plays an essential role in DNA damage repair. It is the least characterized protein of BRCA1-complex and mainly responsible for maintaining the complex integrity. However, its structural and functional aspects of regulating the complex stability still remain elusive. Here, we carried out a comprehensive examination of MERIT40 biophysical properties and identified its novel interacting partner which would help to understand its role in BRCA1-complex. The recombinant protein was purified by affinity chromatography and unfolding pathway was determined using spectroscopic and calorimetric methods. Molecular model was generated using combinatorial approaches of modeling, and monomer-monomer docking was carried out to identify dimeric interface. Disordered region of MERIT40 was hatchet using trypsin and chymotrypsin to illustrate the existence of stable domain whose function was speculated through DALI search. Our findings suggest that MERIT40 forms a dimer in a concentration-independent manner. Its central region shows remarkable stability towards the protease digestion and has structural similarity with vWA-like region, a domain mainly present in complement activation factors. MERIT40 undergoes a three-state unfolding transition pathway with a dimeric intermediate. It interacts with adaptor molecule of BRCA1-complex, called ABRAXAS, thus help in extending the bridging interaction among various members which further stabilizes the whole complex. The results presented in this paper provide first-hand information on structural and folding behavior of MERIT40. These findings will help in elucidating the role of protein-protein interactions in stabilization of BRCA1-complex.

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci.

The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein-ubiquitin interactions.