DNA-damaging Reagents Research Articles

Abstract PO4-28-08: Unraveling the Role of BRCA1-BARD1 E3 Ubiquitin Ligase in DNA Repair: A Promise for Enhanced Chemotherapy Outcomes

Abstract BACKGROUND: BRCA1, a central player in gynecological cancers, collaborates with BARD1 to shape a potent complex pivotal for DNA binding and ubiquitin E3 ligase activities. This complex impacts a myriad of biological pathways. Notably, BRCA1-BARD1’s role in tumor suppression and homology-directed DNA repair (HDR) has been spotlighted. Our innovative approach used RING-domain mutations to craft ligase-dead BRCA1-BARD1 mutants, hypothesizing that these mutants would offer fresh insights into the DNA repair dynamics of BRCA1-BARD1. METHODS: Full length BRCA1-BARD1 or truncated mutants, and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. Stable mammalian cell lines HeLa and MDA-MB-436 that express wild type or mutant forms of BRCA1 and BARD1 were established for a host of analyses, including cellular fractionation, foci analysis, Proximity ligation assay (PLA), comet assays and clonogenic survival assays with various DNA damage agents. RESULTS: By systematic biochemical screening and a series of in vitro assays, we generated a truly BRCA1-BARD1 E3 dead mutant, BRCA1I26A, L63A, K65A-BARD1 (BRCA1-E3d), which lacks E3 ligase activity but possesses all other known attributes such as retention of BRCA1-BARD1 heterodimeric structure formation, DNA binding, and intact RAD51-mediated recombinase activity. To our surprise, we discovered that previously described BRCA1-BARD1 RING-domain mutant (BRCA1-I26A) still possessed ubiquitylation activity not found in BRCA1-E3d. To determine the biological significance of these mutants, cells stably expressing BRCA1-E3d were treated with various DNA-damaging agents and shown to more sensitive than WT or previously identified mutants. DNA repair pathway reporter assays determined these cells were deficient in various repair pathways compared to their BRCA1-WT counterparts. Further studies demonstrate that BRCA1-BARD1 E3 ligase is required for DNA resection during HDR, as evidenced in reduced levels of DNA repair-related foci formation such as RPA, RAD51 and CtIP in BRCA1-E3d cells. Furthermore, compared to BRCA1-WT cells, BRCA1-E3d cells were more sensitive to DNA damage reagents after depletion of 53BP1, which indicates BRCA1-BARD1 E3 ligase function also contributes to later stages of DNA repair completion. CONCLUSIONS: Our work dispels prevailing ambiguities surrounding BRCA1-BARD1 E3 ligase functions, underscoring its paramountcy in genome repair. This trailblazing research not only enriches our understanding but also beckons therapeutic interventions targeting tumor suppression. The unveiling of BRCA1-BARD1 E3 ligase's intricate regulatory dynamics combined with our novel mutants paves the way for an exciting new era in cancer therapeutics, hinting at superior treatments to enhance patient recovery. Citation Format: Wenjing Li, Meiling Wang, Nozomi Tomimatsu, Jae-Hoon Ji, Salvador Alejo, Gangadhara R. Sareddy, Sandeep Burma, Rachel Klevit, Weixing Zhao. Unraveling the Role of BRCA1-BARD1 E3 Ubiquitin Ligase in DNA Repair: A Promise for Enhanced Chemotherapy Outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-08.

Macroscopic inhibition of DNA damage repair pathways by targeting AP-2α with LEI110 eradicates hepatocellular carcinoma

DNA damage repair (DDR) genes are known to be closely associated with the progression of Hepatocellular carcinoma (HCC). Here we report a unique cluster of “deletion-up” genes in HCC, which are accordantly overexpressed in HCC patients and predict the unfavorable prognosis. Binding motif analysis and further validation with ChIP-qPCR unveil that the AP-2α directly modulate the transcription of critical DNA repair genes including TOP2A, NUDT1, POLD1, and PARP1, which facilitates the sanitation of oxidized DNA lesions. Structural analysis and the following validation identify LEI110 as a potent AP-2α inhibitor. Together, we demonstrate that LEI110 stabilizes AP-2α and sensitizes HCC cells toward DNA-damaging reagents. Altogether, we identify AP-2α as a crucial transcription modulator in HCC and propose small-molecule inhibitors targeting AP-2α are a promising novel class of anticancer agents. Our study provides insights into the concept of macroscopic inhibition of DNA damage repair-related genes in cancer treatment.

ATR-binding lncRNA ScaRNA2 promotes cancer resistance through facilitating efficient DNA end resection during homologous recombination repair

BackgroundOur previous study first showed that ATR-binding long noncoding RNA (lncRNA) is necessary for ATR function and promotes cancer resistance. However, the specific lncRNAs instrumental in ATR activation remain largely unclear, which limits our comprehensive understanding of this critical biological process.MethodsRNA immunoprecipitation (RIP) followed by RNA sequencing was employed to identify ATR-binding lncRNAs, which were further validated using RIP-qPCR assays. Immunofluorescence staining and Western blotting were applied to detect the activation of DNA damage repair factors. After the effect of scaRNA2 on cellular sensitivity to DNA-damaging reagents was determined, the effects of scaRNA2 on radiotherapy were investigated in patient-derived organoids and xenograft preclinical models. The clinical relevance of scaRNA2 was also validated in tissues isolated from rectal cancer patients.ResultsScaRNA2 was identified as the most enriched ATR-binding lncRNA and was found to be essential for homologous recombination (HR) mediated DNA damage repair. Furthermore, scaRNA2 knockdown abrogated the recruitment of ATR and its substrates in response to DNA damage. Mechanistically, scaRNA2 was observed to be necessary for Exo1-mediated DNA end resection and bridged the MRN complex to ATR activation. Knockdown of scaRNA2 effectively increased the sensitivity of cancer cells to multiple kinds of DNA damage-related chemoradiotherapy. Preclinically, knockdown of scaRNA2 improved the effects of radiotherapy on patient-derived organoids and xenograft models. Finally, an increase in scaRNA2 colocalized with ATR was also found in clinical patients who were resistant to radiotherapy.ConclusionsScaRNA2 was identified as the most abundant lncRNA bound to ATR and was demonstrated to bridge DNA end resection to ATR activation; thus, it could be applied as a potent target for combined cancer treatments with chemoradiotherapy.

Caffeine enhances chemosensitivity to irinotecan in the treatment of colorectal cancer

Background: Colorectal cancer (CRC) is one of the most common types of cancer. This disease arises from gene mutations and epigenetic alterations that transform colonic epithelial cells into colon adenocarcinoma cells, which display a unique gene expression pattern compared to normal cells. Specifically, CRC cells exhibit significantly higher expression levels of genes involved in DNA repair or replication, which is attributed to the accumulation of DNA breakage resulting from rapid cell cycle progression.Purpose: This study aimed to investigate the in vivo effects of caffeine on CRC cells and evaluate its impact on the sensitivity of these cells to irinotecan, a topoisomerase I inhibitor widely used for CRC treatment.Methods: Two CRC cell lines, HCT116 and HT29, were treated with irinotecan and caffeine. Western blot analysis assessed protein expression levels in caffeine/irinotecan-treated CRC cells. Immunofluorescence staining determined protein localization, measured DNA breaks, and explored the effects of DNA damage reagents during cell cycle progression and flow cytometry analysis was used to measure cell viability. Fiber assays investigated DNA synthesis in DNA-damaged cells during S-phase, while the comet assay assessed DNA fragmentation caused by DNA breaks.Results: Our findings demonstrated that the combination of irinotecan and caffeine exhibits a synergistic effect in suppressing CRC cell proliferation and inducing cell death. Compared to treatment with only irinotecan or caffeine, the combined irinotecan and caffeine treatment was more effective in inducing DNA lesions by displacing RAD51 from DNA break sites and inhibiting DNA repair progression, leading to cell cycle arrest. This combination also resulted in more severe effects, including DNA fragmentation and mitotic catastrophe.Conclusion: Caffeine could enhance the effectiveness of an existing drug for CRC treatment despite having little impact on the cell survival rate of CRC cells. Our findings suggest that the beneficial adjuvant effects of caffeine may not only be applicable to CRC but also to various other types of cancers at different stages of development.

SCARECROW maintains the stem cell niche in Arabidopsis roots by ensuring telomere integrity.

Stem cells are the ultimate source of cells for various tissues and organs and thus are essential for postembryonic plant growth and development. SCARECROW (SCR) is a plant-specific transcription regulator well known for its role in stem cell renewal in plant roots, but the mechanism by which SCR exerts this function remains unclear. To address this question, we carried out a genetic screen for mutants that no longer express SCR in the stem cell niche of Arabidopsis (Arabidopsis thaliana) roots and characterized 1 of these mutants. Molecular genetics methods allowed us to pinpoint the causal mutation in this mutant in TELOMERIC PATHWAYS IN ASSOCIATION WITH STN 1 (TEN1), encoding a factor that protects telomere ends. Interestingly, TEN1 expression was dramatically reduced in the scr mutant. Telomerase and STN1 and CONSERVED TELOMERE MAINTENANCE COMPONENT 1 (CTC1), components of the same protein complex as TEN1, were also dramatically downregulated in scr. Loss of STN1, CTC1, and telomerase caused defects in root stem cells. These results together suggest that SCR maintains root stem cells by promoting expression of genes that ensure genome integrity. Supporting this conclusion, we demonstrated that the scr mutant accumulates more DNA damage than wild-type Arabidopsis and that this problem is aggravated after exposure to zeocin, a DNA damage reagent. Finally, we identified 2 previously uncharacterized motifs in TEN1 and provide evidence that a conserved amino acid residue in 1 of the motifs is indispensable for TEN1 function. SCR thus provides a connection between genome integrity and stem cell maintenance in Arabidopsis roots.

ITGA2 overexpression inhibits DNA repair and confers sensitivity to radiotherapies in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a 5-year survival rate of less than 10%, despite the recent advances in chemoradiotherapy. The sensitivity of the PDAC patients to chemoradiotherapy varies widely, especially to radiotherapy, suggesting the need for more elucidation of the underlying mechanisms. In this study, a novel function of the nuclear ITGA2, the alpha subunit of transmembrane collagen receptor integrin alpha-2/beta-1, regulating the DNA damage response (DDR), was identified. First, analyzing The Cancer Genome Atlas (TCGA) PDAC data set indicated that the expression status of ITGA2 was negatively correlated with the genome stability parameters. The study further demonstrated that ITGA2 specially inhibited the activity of the non-homologous end joining (NHEJ) pathway and conferred the sensitivity to radiotherapy in PDAC by restraining the recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to Ku70/80 heterodimer during DDR. Considering the overexpression of ITGA2 and its associated with the poor prognosis of PDAC patients, this study suggested that the ITGA2 expression status could be used as an indicator for radiotherapy and DNA damage reagents, and the radiotherapy in combination with the overexpression of ITGA2 might be a viable treatment strategy for the PDAC patients.

A plant-specific module for homologous recombination repair

Homologous recombination repair (HR) is an error-free DNA damage repair pathway to maintain genome stability and a basis of gene targeting using genome-editing tools. However, the mechanisms of HR in plants are still poorly understood. Through genetic screens for DNA damage response mutants (DDRM) in Arabidopsis, we find that a plant-specific ubiquitin E3 ligase DDRM1 is required for HR. DDRM1 contains an N-terminal BRCT (BRCA1 C-terminal) domain and a C-terminal RING (really interesting new gene) domain and is highly conserved in plants including mosses. The ddrm1 mutant is defective in HR and thus is hypersensitive to DNA-damaging reagents. Biochemical studies reveal that DDRM1 interacts with and ubiquitinates the transcription factor SOG1, a plant-specific master regulator of DNA damage responses. Interestingly, DDRM1-mediated ubiquitination promotes the stability of SOG1. Consistently, genetic data support that SOG1 functions downstream of DDRM1. Our study reveals that DDRM1-SOG1 is a plant-specific module for HR and highlights the importance of ubiquitination in HR.

Microfluidic chip-based long-term preservation and culture of engineering bacteria for DNA damage evaluation.

Understanding the effects of long-term exposure to space environment is paramount to maintaining the safety, health of astronauts. The physical dosimeters currently used on the space station cannot be used to assess the physiological effects of radiation. Moreover, some developed biological methods are time-consuming and passive and cannot be used for active and real-time detection of the physiological effects of radiation in space environment. Here, the SOS promoter: recA-eGFP genetic engineering bacteria was constructed and characterized, and DNA damage effects of some chemical reagents and radiation were evaluated. The results indicated the constructed engineering bacteria can distinguish DNA damage reagents from non-damage reagents and have a good dose-fluorescence effect against Co-60 radiation with the detection limit of 0.64Gy; in order to overcome the restriction of long-term preservation of bacteria in space environment, the bacteria were freeze-dried, and the protectants were optimized, the storage time of bacteria under dry conditions was explored by accelerated storage experiment. Finally, a microfluidic chip was designed and fabricated for freeze-drying genetic engineering bacteria recovery, culture, and analysis in space environment. This study can provide support for the establishment of on-orbit radiation damage risk monitoring and early warning and can provide basic data for maintaining the health and performance of astronauts on long-term space flight missions. Moreover, the technique developed herein has a great potential to be used as a powerful tool for efficiently screening various radioactive substance, toxic chemicals, drugs, etc. KEY POINTS: • The SOS promoter: recA-eGFP genetic engineering bacteria was successfully constructed, which can distinguish DNA damage reagents from non-damage reagents and possess a good dose-effect relationship against Co-60 radiation. • The bacteria were freeze-dried to overcome the restriction of long-term preservation of bacteria in space environment, and protectants were optimized, and the survival rate of freeze-dried engineering bacteria can be predicted based on the results of accelerated storage experiment. • Microfluidic chip-based culture platform was successfully designed, fabricated, and used for freeze-drying genetic engineering bacteria recovery, culture, and analysis.

Overexpression of 15-Hydroxyprostaglandin Dehydrogenase Inhibits A549 Lung Adenocarcinoma Cell Growth via Inducing Cell Cycle Arrest and Inhibiting Epithelial-Mesenchymal Transition.

PurposeLung cancer is one of the most commonly diagnosed cancer as well as the leading cause of cancer-related mortality worldwide, among which lung adenocarcinoma (LUAD) is the most frequent form of lung cancer. Previous studies have shown that 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of prostaglandins to reduce their biological activities and behaves as a tumor suppressor in various cancers. Thus, we aimed to systematically examine the effects of 15-PGDH overexpression on cellular processes in lung adenocarcinoma cells.MethodsThe stable 15-PGDH-overexpressing A549 cell line was constructed using lentivirus particles. CCK-8 assay was used to determine the cell proliferation rate and sensitivity to cisplatin. Tandem mass tag (TMT)-based quantitative proteomic analysis was used to identify differentially expressed proteins between control and 15-PGDH-overexpression cells. The cell cycle was determined by a flow cytometer. The expression levels of mesenchymal and epithelial markers were measured using Western blotting. Wound healing and transwell assays were used to detect the cell migration and cell invasion ability, respectively.ResultsAnalysis of datasets in The Cancer Genome Atlas revealed that the PGDH gene expression level in the lung adenocarcinoma tissues was significantly lower than that in the pericarcinous tissues. 15-PGDH overexpression in A549 cells reduced cell proliferation rate. Quantitative proteomics revealed that 15-PGDH overexpression inhibited PI3K/AKT/mTOR signaling pathway, which is a signaling pathway driving tumor cell growth and epithelial-mesenchymal transition (EMT) process. In addition, both cell cycle and DNA repair-related proteins were down-regulated in 15-PGDH overexpressed cells. 15-PGDH overexpression induced G1/S cell cycle arrest and increased susceptibility to DNA damaging reagent cisplatin. Importantly, overexpression of 15-PGDH inhibited EMT process with the downregulation of β-catenin and Snail-1 as well as upregulation of E-cadherin and ZO-1.Conclusion15-PGDH is a tumor suppressor in lung cancer and may serve as a potential therapeutic target to prevent lung adenocarcinoma.

Bacterial YedK represses plasmid DNA replication and transformation through its DNA single-strand binding activity

The SOS response-associated peptidase (SRAP) is an ancient protein superfamily in all domains of life. The mammalian SRAP was recently reported to covalently bind to the abasic sites (AP) in single stranded (ss) DNA to shield the chromosome integrity. YedK, the Escherichia coli SRAP, is not functionally characterized. Here we report the fortuitous pull-down of YedK from bacterial cell lysates by short (<20 bp) double stranded (ds) DNAs, further enrichment of YedK was observed when single stranded (ss) DNA was added. YedK can bind multiple DNA substrates, particularly with a high affinity to DNA duplex with single strand segment. As a SRAP protein, the involvement of YedK in SOS response was extensively examined, however yedK mutant of Escherichia coli showed no difference from the wild type strain upon the treatments with UV and various DNA damaging reagents, indicating its non-essentiality or redundancy in E. coli. Surprisingly, yedK mutants derived from Escherichia coli and Samonella enterica both showed an increased plasmid DNA transformation efficiency compared to the wild types. In accordance with this, induction of YedK effectively decreased the copy number of plasmid DNA. Site-directed mutagenesis of YedK demonstrated that residues involved in single strand DNA binding and cysteine residue at position 2 from N-terminus can discharge the repression of the plasmid transformation efficiency.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

A CRISPR-associated factor Csa3a regulates DNA damage repair in Crenarchaeon Sulfolobus islandicus.

CRISPR−Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability.

Potential use of homologous recombination deficiency score as an indicator of therapy selection in leiomyosarcoma patients.

e23539 Background: Leiomyosarcoma (LMS) is one of the most frequent sarcoma subtypes. While surgical resection is the standard treatment of LMS, chemotherapy and radiation have shown additional survival benefit. However, the treatment response is variable and selection of appropriate agents is difficult due to the great heterogeneity of LMS. Matching DNA damaging reagents or PARP inhibitors, to tumors with a general homologous recombination deficit (HRD), can help to deploy these reagents more precisely. We explored the correlation of HRD score with the activation of PARP signaling pathway and retrospectively tested the effect of platinum-based therapy on an LMS patient with high HRD score. Methods: Nineteen LMS samples were subjected to whole exome sequencing and their HRD scores were calculated using a published algorithm. Nine of the LMS samples were also subjected to RNA-seq analysis and undergone GSEA to profile PARP pathway expression. CIBERSORT was employed to profile infiltrating immune cells. Results: The 19 LMS samples are defined as HRD-high (11 samples) or HRD-low (8 samples) if their HRD score is above or equal to, or below the medium respectively. For the 9 samples undergone RNA-seq, 97 genes have differential expression between the two groups (p-value &lt; 0.05, fold change≥ 2). GSEA revealed 23 enriched pathways. Interestingly, PPAR signaling pathway is the most highly enriched one (EnrichmentScore = 0.506, P.adj = 0.005). No other pathogenic germline or somatic mutation in HRD related genes, for example BRCA1/2, is enriched. Additionally, high HRD score is significantly correlated with infiltrating Tregs, monocyte, and M0 macrophages as found in CIBERSORT analysis. To retrospectively test whether HRD score can be used to predict the response to inhibitors of DNA repair pathway, we identified one patient who had received platinum-based combination therapy after failure of first-line chemotherapy. The patient is a 40-year-old female with stage IV LMS and has a high HRD score. She achieved PR with 6-month PFS and remains progression free up to the abstract date, supporting a good correlation between high HRD score and advanced chemotherapy in LMS patients. Conclusions: High HRD score is correlated with activated PPAR pathway and infiltrating Tregs, monocytes and M0 macrophages, and can potentially be used to identify LMS patients with good response to platinum-based chemotherapy even though they have no obvious germline and somatic mutation in genes involved in DNA repair.

BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites.

Long non-coding RNAs (lncRNAs) are emerging regulators of genomic stability and human disease. However, the molecular mechanisms by which nuclear lncRNAs directly contribute to DNA damage responses remain largely unknown. Using RNA antisense purification coupled with quantitative mass spectrometry (RAP-qMS), we found that the lncRNA BGL3 binds to PARP1 and BARD1, exhibiting unexpected roles in homologous recombination. Mechanistically, BGL3 is recruited to DNA double-strand breaks (DSBs) by PARP1 at an early time point, which requires its interaction with the DNA-binding domain of PARP1. BGL3 also binds the C-terminal BRCT domain and an internal region (amino acids 127-424) of BARD1, which mediates interaction of the BRCA1/BARD1 complex with its binding partners such as HP1γ and RAD51, resulting in BRCA1/BARD1 retention at DSBs. Cells depleted for BGL3 displayed genomic instability and were sensitive to DNA-damaging reagents. Overall, our findings underscore the biochemical versatility of RNA as a mediator molecule in the DNA damage response pathway, which affects the accumulation of BRCA1/BARD1 at DSBs.

Wdr70 regulates histone modification and genomic maintenance in fission yeast

Eukaryotic genomes are packaged into highly condensed chromatin and this repressive chromatin barrier can be overcome by altering the chromatin structure via histone modification enzymes. Here, we report Wdr70 in Schizosaccharomyces pombe (spWdr70) plays important roles in multiple cellular processes including cell cycle progression, chromatin structure and DNA repair. Depletion of Wdr70 gene causes cell cycle delay, hypersensitivity to DNA damage reagents and quick phenotypic changes. Moreover, we observed strong genetic interaction between Wdr70 and genes regulating checkpoint and homologous recombination (HR), pinpointing the function of Wdr70 to DNA end resection. Finally, we show that the function of Wdr70 could be attributed to monoubiquitination of histone H2B (uH2B) in the vicinity of DNA double strand breaks (DSBs). Taken together, our data reveal that Wdr70 and H2B monoubiquitination-dependent chromatin modulation is required for chromatin homeostasis and genetic stability.

PRKDC is a prognostic marker for poor survival in gastric cancer patients and regulates DNA damage response

A hallmark of gastric cancer is the high rate of genomic instability associated with deregulation of DNA damage repair pathways. DNA-Dependent Protein Kinase Catalytic Subunit (PRKDC) is a key component of the non-homologous end-joining (NHEJ) pathway. By reanalyzing transcriptome data of 80 pairs of gastric cancer tumors and the adjacent normal tissues from non-treated patients, we identified PRKDC as the top upregulated DNA damage repair genes in gastric cancer. High expression of PRKDC is associated with poor survival of gastric cancer patients, and genomic amplification of the gene is frequently observed across most gastric cancer subtypes. Knockdown of PRKDC in gastric cell lines resulted in reduced proliferation and cell cycle arrest. Furthermore, we showed that loss of PRKDC induced DNA damage and enhanced gastric cancer cell chemosensitivity to DNA-damaging reagents. Together, our results suggest that PRKDC is a prognostic marker of poor survival and is a putative target to overcome chemoresistance in gastric cancer.

PF575 OVEREXPRESSION DNP73 PROMOTES THE PROLIFERATION AND DRUG RESISTANCE OF MULTIPLE MYELOMA

Background:Genomic instability and chromosomal abnormalities induced by DNA damage are important biological characteristics of multiple myeloma (MM). DNp73 is one of the main isomers of TP73, a member of the TP53 protein family. We found that DNp73 was highly expressed in MM which could resist the apoptosis induced by DNA damage in MM cells, leading to clinical drug resistance.Aims:In order to further explore the mechanism of DNp73 mediating drug resistance to DNA damage in MM, this study verified and explored from multiple levels including cell phenotype changes in vivo and in vitro.Methods:MM cell lines OCI‐MY5 and ARP1 were used to constructed DNp73 overexpression (OE) cell lines, respectively. Cell proliferation activity of DNp73 OE and EV (empty vector) cell lines was compared by absolute cell count method and CCK8 method, and cell proliferation was determined under the action of doxorubicin (5nM &amp;50 nM) or epirubicin (5nM &amp; 10 nM) at different concentrations. The cloning formation and drug sensitivity of ARP1 DNp73 OE/EV cells were determined by soft agar cloning formation assay. Flow cytometry was used to detect the apoptosis and cell cycle arrest of cells in DNp73 OE and EV cells under UV and at different concentrations of reagents treatment. Xenografted model and 5TGM1 spontaneous mouse MM model were used to detect the effect of DNp73 on the proliferation of MM cells and the treatment of DNA damage reagents in vivo, as well as the survival period of tumor‐bearing mice were analyzed. RNA‐seq and gene enrichment analysis (GSEA) were performed to explore the potential signaling pathway of DNp73 against apoptosis. Further western blotting and quantity‐real time PCR were performed for validation. ChIP and CoIP were used to search for the potential downstream key target genes and target proteins of DNp73.Results:DNp73‐OE ARP1 &amp; OCI‐MY5 cell lines showed stronger proliferation activity than EV MM cell lines, and showed significant resistance to DNA damage reagents, doxorubicin and epirubicin treatment. The cloning formation experiment of soft agar confirmed that DNp73 induced the resistance of MM cells to those drugs. Flow cytometry analysis showed that DNp73 OE suppressed the MM cells apoptosis and suppressed G1 phase arrest induced by those drugs treatment (P &lt; 0.05). In vivo experiments showed that the tumor burden of mice was significantly more severe compared with the mice of control group. The tumor‐bearing mice in DNp73 OE group were resistant to the treatment of DNA damage reagents. Moreover, the survival of DNp73 OE tumor‐bearing mice was significantly shortened compared with the control group. RNA‐seq, GSEA analysis and western blot assay revealed that DNp73 overexpression induce drug resistance and proliferation through the activation of c‐myc and AKT‐related pathways and inhibition of p21/p27 signal pathway which caused cell cycle arrested in MM.Summary/Conclusion:DNp73 promote MM cell proliferation, anti‐apoptosis and inhibit cell cycle arrest through activating c‐myc and Akt‐related pathways. DNp73 overexpression is related to the occurrence of drug resistance in MM.image

Dissecting PCNA function with a systematically designed mutant library in yeast

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sensitivity to DNA damaging reagents were identified using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway.

MDM2-mediated degradation of WRN promotes cellular senescence in a p53-independent manner.

MDM2 (Murine double minute 2) acts as a key repressor for p53-mediated tumor-suppressor functions, which includes cellular senescence. We found that MDM2 can promote cellular senescence by modulating WRN stability. Werner syndrome (WS), caused by mutations of the WRN gene, is an autosomal recessive disease, which is characterized by premature aging. Loss of WRN function induces cellular senescence in human cancer cells. Here, we found that MDM2 acts as an E3 ligase for WRN protein. MDM2 interacts with WRN both in vivo and in vitro. MDM2 induces ubiquitination of WRN and dramatically downregulates the levels of WRN protein in human cells. During DNA damage response, WRN is translocated to the nucleoplasm to facilitate its DNA repair functions; however, it is degraded by the MDM2-mediated ubiquitination pathway. Moreover, the senescent phenotype induced by DNA damage reagents, such as Etoposide, is at least in part mediated by MDM2-dependent WRN degradation as it can be significantly attenuated by ectopic expression of WRN. These results show that MDM2 is critically involved in regulating WRN function via ubiquitin-dependent degradation and reveal an unexpected role of MDM2 in promoting cellular senescence through a p53-independent manner.

The trimeric Hef-associated nuclease HAN is a 3'→5' exonuclease and is probably involved in DNA repair.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN’s function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3′-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3′-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.