Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-Stranded RNA

3\u27 repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent Km of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1

Role of FANCA in DNA Damage Repair

Fanconi anemia (FA) is a severe genetic disorder characterized by bone marrow failure, developmental defects, chromosomal instability, and predisposition to cancer. FA cells are hypersensitive to DNA crosslinking compounds including mitomycin C (MMC), cisplatin, and diepoxybutane, which make defective interstrand crosslink (ICL) repair mechanisms a hallmark for the disease. Of the 17 genes associated with FA, mutations in the complementation group A (FANCA) account for ~64% of all patient cases. Studies explored in this dissertation comprise the biochemical details for the functional role of FANCA in DNA ICL repair and the first biochemical evidence for its possible role in double-strand break (DSB) repair. In an oligonucleotide-based assay purified FANCA shows enhancement of MUS81- EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of ICLs. A prevalent hypothetical role of MUS81-EME1 in ICL repair was to unhook the damage by incising the leading strand on the 3’ side of an ICL lesion. The studies presented in Chapter 3 show that purified MUS81-EME1 incises DNA on the 5’ side of a psoralen ICL residing in fork-like structures. Using truncated FANCA proteins, I determined that both the N- and C-regions of the protein are required for the observed FANCA-dependent MUS81-EME1 incision regulation. Using laser-induced psoralen ICL formation in cells, I found that FANCA colocalizes with and recruits MUS81 to ICL lesions. Due to the specific ICL recognition activity of FANCA in vitro, I hypothesized that it was likely to catalyze strand separation in order to discriminate damage that crosslinks the DNA duplex from damage that only affects one strand. To test my hypothesis, I examined the effect of FANCA on DNA stability. In an oligonucleotide-based assay purified FANCA shows strong helix destabilization, single-strand annealing and strand exchange activities that are completely dependent on protein:oligonucleotide stoichiometric ratios. While low stoichiometric ratios of FANCA to DNA result in helix destabilization, higher ratios catalyze single-strand annealing and strand exchange. Furthermore, FANCA and RAD51 exhibit synergistic DNA strand annealing activity, suggesting their coordinated function might play a role in fork stability. FANCA also promotes the bidirectional annealing of structures that mimic physiologically relevant intermediates in DSB repair. C- and N-terminal truncation mutants reveal that binding to DNA is not sufficient for the helix destabilization or single-strand annealing activities of FANCA. Patient-derived FANCA mutants Q772X, D598N, R1117G, Q1128E, and F1263Δ exhibit deregulated helix destabilization, strand annealing and strand exchange activities. It is conceivable that proteins harboring helix destabilization, single-strand annealing, and strand exchange activities work in concert to protect genome integrity and efficiently process replication and recombination intermediate structures. It’s possible that in the presence of an ICL, FANCA may serve to recognize the damage, help anchor the FA core complex to the DNA, recruit structure-specific endonuclease MUS81/EME1 and enhance its incision activity accordingly. When replication forks are stalled due to sources other than ICLs, FANCA may be recruited to remodel the fork and promote fork- restart through ssDNA annealing of excessively unwound template or fork regression and “chicken foot” formation through single-strand annealing of the nascent DNA. FANCA may also act as a local helix destabilizing protein that promotes fork protection by drawing the nuclease away from the fork, thereby inhibiting it’s incision activity. Collectively, it appears FANCA may have multiple roles in ICL repair, particularly downstream of FA pathway activation during the repair of intermediate DSB structures. It’s possible that FANCA works in concert with RAD51 or RAD52 to promote the repair of DSBs through homology-directed pathways strand invasion and single-strand annealing.</p

Dietary supplement hymecromone and sorafenib: A novel combination for the control of renal cell carcinoma

Purpose Current treatments for metastatic renal cell carcinoma do not extend survival beyond a few months. Sorafenib is a targeted drug approved for metastatic renal cell carcinoma but it has modest efficacy. Hymecromone is a nontoxic dietary supplement with some antitumor activity at high doses of 450 to 3,000 mg per day. Hymecromone inhibits the synthesis of hyaluronic acid, which promotes tumor growth and metastasis. We recently noted that the hyaluronic acid receptors CD44 and RHAMM are potential predictors of metastatic renal cell carcinoma. In the current study we examined the antitumor properties of hymecromone, sorafenib and the combination in renal cell carcinoma models. Materials and Methods Using proliferation, clonogenic and apoptosis assays, we examined the effects of hymecromone (0 to 32 μg/ml), sorafenib (0 to 3.2 μg/ml) and hymecromone plus sorafenib in Caki-1, 786-O, ACHN and A498 renal cell carcinoma cells, and HMVEC-L and HUVEC endothelial cells. A Boyden chamber was used for motility and invasion assays. Apoptosis indicators, hyaluronic acid receptors, epidermal growth factor receptor and c-Met were evaluated by immunoblot. The efficacy of hymecromone, sorafenib and hymecromone plus sorafenib was assessed in the sorafenib resistant Caki-1 xenograft model. Results Hymecromone plus sorafenib synergistically inhibited proliferation (greater than 95%), motility/invasion (65%) and capillary formation (76%) in renal cell carcinoma and/or endothelial cells, and induced apoptosis eightfold (p <0.001). Hymecromone plus sorafenib inhibited hyaluronic acid synthesis and adding hyaluronic acid reversed the cytotoxicity of hymecromone plus sorafenib. Hymecromone plus sorafenib up-regulated pro-apoptotic indicators and down-regulated Mcl-1, CD44, RHAMM, phospho-epidermal growth factor receptor and phospho-cMet. In all assays hymecromone and sorafenib alone were ineffective. Oral administration of hymecromone (50 to 200 mg/kg) plus sorafenib (30 mg/kg) eradicated Caki-1 tumor growth without toxicity. Hymecromone and sorafenib alone were ineffective. Conclusions To our knowledge this is the first study to show that the combination of sorafenib and the nontoxic dietary supplement hymecromone is highly effective for controlling renal cell carcinoma

Abstract 39: FANCA regulates MUS81-EME1 mediated DNA incision in a damage-dependent manner

Abstract MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand crosslinks (ICL). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, interstrand crosslink repair protein, FANCA, greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner. Citation Format: Anaid Benitez, Fenghua Yuan, Satoshi Nakajima, Leizhen Wei, Liangyue Qian, Richard Myers, Jennifer J. Hu, Li Lan, Yanbin Zhang. FANCA regulates MUS81-EME1 mediated DNA incision in a damage-dependent manner. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 39. doi:10.1158/1538-7445.CANSUSC14-39</jats:p

Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA

3′ repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent Km of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1. Background: 3′ repair exonuclease 1 (TREX1) is a DNase involved in autoimmune disorders and the antiviral response. Results: TREX1 also degrades single-stranded RNA or RNA in a RNA/DNA hybrid molecule. Conclusion: TREX1 is a human homolog of Escherichia coli RNase T. Significance: The novel RNase activity of TREX1 is crucial for understanding its physiological role

Targeting Hyaluronidase for Cancer Therapy: Antitumor Activity of Sulfated Hyaluronic Acid in Prostate Cancer Cells

The tumor cell-derived hyaluronidase HYAL-1 degrades hyaluronic acid (HA) into pro-angiogenic fragments that support tumor progression. Although HYAL-1 is a critical determinant of tumor progression and a marker for cancer diagnosis and metastasis prediction, it has not been evaluated as a target for cancer therapy. Similarly, sulfated hyaluronic acid (sHA) has not been evaluated for biological activity, although it is a HAase inhibitor. In this study we show that sHA is a potent inhibitor of prostate cancer. sHA blocked the proliferation, motility and invasion of LNCaP, LNCaP-AI, DU145 and LAPC-4 prostate cancer cells, also inducing caspase 8-dependent apoptosis associated with downregulation of Bcl-2 and phospho-Bad. sHA inhibited Akt signaling including androgen receptor (AR) phosphorylation, AR-activity, NFkb activation and VEGF expression. These effects were traced to a blockade in complex formation between PI3K and HA receptors and to a transcriptional downregulation of HA receptors, CD44 and RHAMM, along with PI3K inhibition. Angiogenic HA fragments or overexpression of myristoylated-Akt or HA receptors blunted these effects of sHA, implicating a feedback loop between HA receptors and PI3K/Akt signaling in the mechanism of action. In an animal model, sHA strongly inhibited LNCaP-AI prostate tumor growth without causing weight loss or apparent serum-organ toxicity. Inhibition of tumor growth was accompanied by a significant decrease in tumor angiogenesis and an increase in apoptosis index. Taken together, our findings offer mechanistic insights into the tumor-associated HA-HAase system and a preclinical proof-of-concept of the safety and efficacy of sHA to control prostate cancer growth and progression

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genomewide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutationassociated chromosome fragility and AOA2

FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange

FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52. [Display omitted] •FANCA catalyzes bidirectional single-strand annealing and strand exchange•FANCG stimulates FANCA-mediated strand annealing and strand exchange•Fanconi anemia patient-derived FANCA mutants are deficient in both activities•The single-strand annealing activity of FANCA plays a direct role in DSB repair Benitez et al. report that FANCA biochemically catalyzes single-strand annealing and strand exchange. They find that the single-strand annealing activity of FANCA is relevant to the etiology of Fanconi anemia and responsible for its involvement in double-strand break repair, which is independent of the canonical FA pathway and RAD52

Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner