Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Targeting HR Repair as a Synthetic Lethal Approach to Increase DNA Damage Sensitivity by a RAD52 Inhibitor in BRCA2-Deficient Cancer Cells.

BRCA mutation, one of the most common types of mutations in breast and ovarian cancer, has been suggested to be synthetically lethal with depletion of RAD52. Pharmacologically inhibiting RAD52 specifically eradicates BRCA-deficient cancer cells. In this study, we demonstrated that curcumin, a plant polyphenol, sensitizes BRCA2-deficient cells to CPT-11 by impairing RAD52 recombinase in MCF7 cells. More specifically, in MCF7-siBRCA2 cells, curcumin reduced homologous recombination, resulting in tumor growth suppression. Furthermore, a BRCA2-deficient cell line, Capan1, became resistant to CPT-11 when BRCA2 was reintroduced. In vivo, xenograft model studies showed that curcumin combined with CPT-11 reduced the growth of BRCA2-knockout MCF7 tumors but not MCF7 tumors. In conclusion, our data indicate that curcumin, which has RAD52 inhibitor activity, is a promising candidate for sensitizing BRCA2-deficient cells to DNA damage-based cancer therapies.

Yeast Rpn4 Links the Proteasome and DNA Repair via RAD52 Regulation.

Environmental and intracellular factors often damage DNA, but multiple DNA repair pathways maintain genome integrity. In yeast, the 26S proteasome and its transcriptional regulator and substrate Rpn4 are involved in DNA damage resistance. Paradoxically, while proteasome dysfunction may induce hyper-resistance to DNA-damaging agents, Rpn4 malfunction sensitizes yeasts to these agents. Previously, we proposed that proteasome inhibition causes Rpn4 stabilization followed by the upregulation of Rpn4-dependent DNA repair genes and pathways. Here, we aimed to elucidate the key Rpn4 targets responsible for DNA damage hyper-resistance in proteasome mutants. We impaired the Rpn4-mediated regulation of candidate genes using the CRISPR/Cas9 system and tested the sensitivity of mutant strains to 4-NQO, MMS and zeocin. We found that the separate or simultaneous deregulation of 19S or 20S proteasome subcomplexes induced MAG1, DDI1, RAD23 and RAD52 in an Rpn4-dependent manner. Deregulation of RAD23, DDI1 and RAD52 sensitized yeast to DNA damage. Genetic, epigenetic or dihydrocoumarin-mediated RAD52 repression restored the sensitivity of the proteasome mutants to DNA damage. Our results suggest that the Rpn4-mediated overexpression of DNA repair genes, especially RAD52, defines the DNA damage hyper-resistant phenotype of proteasome mutants. The developed yeast model is useful for characterizing drugs that reverse the DNA damage hyper-resistance phenotypes of cancers.

Human RECQL4 represses the RAD52-mediated single-strand annealing pathway after ionizing radiation or cisplatin treatment.

Ionizing radiation (IR) and cisplatin are frequently used cancer treatments, although the mechanisms of error-prone DNA repair-mediated genomic instability after anticancer treatment are not fully clarified yet. RECQL4 mutations mainly in the C-terminal region of the RECQL4 gene lead to the cancer-predisposing Rothmund-Thomson syndrome, but the function of RECQL4ΔC (C-terminus deleted) in error-prone DNA repair remains unclear. We established several RECQL4ΔC cell lines and found that RECQL4ΔC cancer cells, but not RECQL4ΔC nontumorigenic cells, exhibited IR/cisplatin hypersensitivity. Notably, RECQL4ΔC cancer cells presented increased RPA2/RAD52 foci after cancer treatments. RECQL4ΔC HCT116 cells exhibited increased error-prone single-strand annealing (SSA) activity and decreased alternative end-joining activities, suggesting that RECQL4 regulates the DNA repair pathway choice at double-strand breaks. RAD52 depletion by siRNA or RAD52 inhibitors (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside [AICAR], (-)-epigallocatechin [EGC]) or a RAD52-phenylalanine 79 aptamer significantly restrained the growth of RAD52-upregulated RECQL4ΔC HCT116 cells in vitro and in mouse xenografts. Remarkably, compared to single-agent cisplatin or EGC treatment, cisplatin followed by low-concentration EGC had a significant suppressive effect on RECQL4ΔC HCT116 cell growth in vivo. Together, the regimens targeting the RAD52-mediated SSA pathway after anticancer treatment may be applicable for cancer patients with RECQL4 gene mutations.

The concerted roles of FANCM and Rad52 in the protection of common fragile sites.

Common fragile sites (CFSs) are prone to chromosomal breakage and are hotspots for chromosomal rearrangements in cancer cells. We uncovered a novel function of Fanconi anemia (FA) protein FANCM in the protection of CFSs that is independent of the FA core complex and the FANCI-FANCD2 complex. FANCM, along with its binding partners FAAP24 and MHF1/2, is recruited to CFS-derived structure-prone AT-rich sequences, where it suppresses DNA double-strand break (DSB) formation and mitotic recombination in a manner dependent on FANCM translocase activity. Interestingly, we also identified an indispensable function of Rad52 in the repair of DSBs at CFS-derived AT-rich sequences, despite its nonessential function in general homologous recombination (HR) in mammalian cells. Suppression of Rad52 expression in combination with FANCM knockout drastically reduces cell and tumor growth, suggesting a synthetic lethality interaction between these two genes, which offers a potential targeted treatment strategy for FANCM-deficient tumors with Rad52 inhibition.

Simultaneous Targeting of PARP1 and RAD52 Triggers Dual Synthetic Lethality in BRCA-Deficient Tumor Cells.

PARP inhibitors (PARPis) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success. We hypothesized that RAD52-mediated DNA repair remains active in PARPi-treated BRCA-deficient tumor cells and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show that RAD52 inhibitors (RAD52is) attenuated single-strand annealing (SSA) and residual homologous recombination (HR) in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with inhibitors or dominant-negative mutants caused synergistic accumulation of DSBs and eradication of BRCA-deficient but not BRCA-proficient tumor cells. Remarkably, Parp1-/-;Rad52-/- mice are normal and display prolonged latency of BRCA1-deficient leukemia compared with Parp1-/- and Rad52-/- counterparts. Finally, PARPi+RAD52i exerted synergistic activity against BRCA1-deficient tumors in immunodeficient mice with minimal toxicity to normal cells and tissues. In conclusion, our data indicate that addition of RAD52i will improve therapeutic outcome of BRCA-deficient malignancies treated with PARPi.

Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52.

Effective DNA repair enables cancer cells to survive DNA damage induced by chemotherapeutic or radiotherapeutic treatments. Therefore, inhibiting DNA repair pathways is a promising therapeutic strategy for increasing the efficacy of such treatments. In this study, we found that dihydrocoumarin (DHC), a flavoring agent, causes deficiencies in double-stand break (DSB) repair and prolonged DNA damage checkpoint recovery in yeast. Following DNA damage, Rad52 recombinase was revealed to be inhibited by DHC, which results in deficiencies in DSB repair and prolonged DNA damage checkpoint recovery. The deletion of RPD3, a class I histone deacetylase (HDAC), was found to mimic DHC-induced suppression of Rad52 expression, suggesting that the HDAC inhibitor activity of DHC is critical to DSB repair and DNA damage sensitivity. Overall, our findings delineate the regulatory mechanisms of DHC in DSB repair and suggest that it might potentially be used as an inhibitor of the DNA repair pathway in human cells.

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase.

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.

Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors.

RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.

Small-Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA-Deficient Cancers.

Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers.

Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile.

Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

DNA repair and synthetic lethality.

Tumors often have DNA repair defects, suggesting additional inhibition of other DNA repair pathways in tumors may lead to synthetic lethality. Accumulating data demonstrate that DNA repair-defective tumors, in particular homologous recombination (HR), are highly sensitive to DNA-damaging agents. Thus, HR-defective tumors exhibit potential vulnerability to the synthetic lethality approach, which may lead to new therapeutic strategies. It is well known that poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) inhibitors show the synthetically lethal effect in tumors defective in BRCA1 or BRCA2 genes encoded proteins that are required for efficient HR. In this review, we summarize the strategies of targeting DNA repair pathways and other DNA metabolic functions to cause synthetic lethality in HR-defective tumor cells.

Rad51 protein controls Rad52-mediated DNA annealing.

In Saccharomyces cerevisiae, Rad52 protein plays an essential role in the repair of DNA double-stranded breaks (DSBs). Rad52 and its orthologs possess the unique capacity to anneal single-stranded DNA (ssDNA) complexed with its cognate ssDNA-binding protein, RPA. This annealing activity is used in multiple mechanisms of DSB repair: single-stranded annealing, synthesis-dependent strand annealing, and cross-over formation. Here we report that the S. cerevisiae DNA strand exchange protein, Rad51, prevents Rad52-mediated annealing of complementary ssDNA. Efficient inhibition is ATP-dependent and involves a specific interaction between Rad51 and Rad52. Free Rad51 can limit DNA annealing by Rad52, but the Rad51 nucleoprotein filament is even more effective. We also discovered that the budding yeast Rad52 paralog, Rad59 protein, partially restores Rad52-dependent DNA annealing in the presence of Rad51, suggesting that Rad52 and Rad59 function coordinately to enhance recombinational DNA repair either by directing the processed DSBs to repair by DNA strand annealing or by promoting second end capture to form a double Holliday junction. This regulation of Rad52-mediated annealing suggests a control function for Rad51 in deciding the recombination path taken for a processed DNA break; the ssDNA can be directed to either Rad51-mediated DNA strand invasion or to Rad52-mediated DNA annealing. This channeling determines the nature of the subsequent repair process and is consistent with the observed competition between these pathways in vivo.