DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in ...adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.
DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several ...neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. ...The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.
Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels ...and signaling modulate inflammation in diabetic kidneys.
Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)(536).
Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser(536) phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding.
These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB-linked inflammatory proteins via a mechanism that involves phosphorylation of Ser(536) in the transactivation domain of RelA.
How huntingtin (HTT) triggers neurotoxicity in Huntington's disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A ...(POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.
RNA is an important target for chemical probes of function and lead therapeutics; however, it is difficult to target with small molecules. One approach to tackle this problem is to identify compounds ...that target RNA structures and utilize them to multivalently target RNA. Here we show that small molecules can be identified to selectively bind RNA base pairs by probing a library of RNA-focused small molecules. A small molecule that selectively binds AU base pairs informed design of a dimeric compound (2AU-2) that targets the pathogenic RNA, expanded r(AUUCU) repeats, that causes spinocerebellar ataxia type 10 (SCA10) in patient-derived cells. Indeed, 2AU-2 (50 nM) ameliorates various aspects of SCA10 pathology including improvement of mitochondrial dysfunction, reduced activation of caspase 3, and reduction of nuclear foci. These studies provide a first-in-class chemical probe to study SCA10 RNA toxicity and potentially define broadly applicable compounds targeting RNA AU base pairs in cells.
Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein substrates in order to alter their degradation rate, sub-cellular ...localization, interaction, and activity. The induction of apoptosis upon USP7 inhibition is well established in cancer containing wild type p53, which operates through the 'USP7-Mdm2-p53' axis. However, in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7 inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7 correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting apoptosis.
Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing ...question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans.
Background: NEIL2 (Nei-like 2) is a mammalian oxidized base-specific DNA glycosylase.
Results:Neil2-null mice accumulate oxidative damage in transcribed genes and are susceptible to inflammatory agents.
Conclusion: In long-lived species, NEIL2 plays a critical role in maintaining genomic integrity and tissue homeostasis.
Significance: We provide in vivo evidence for NEIL2's role in preferential repair of oxidized bases in active genes in mammals.
Preferential repair of bulky DNA adducts from the transcribed genes via nucleotide excision repair is well characterized in mammalian cells. However, definitive evidence is lacking for similar repair ...of oxidized bases, the major endogenous DNA lesions. Here we show that the oxidized base-specific human DNA glycosylase NEIL2 associates with RNA polymerase II and the transcriptional regulator heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), both in vitro and in cells. NEIL2 immunocomplexes from cell extracts preferentially repaired the mutagenic cytosine oxidation product 5-hydroxyuracil in the transcribed strand. In a reconstituted system, we also observed NEIL2-initiated transcription-dependent base excision repair of 5-hydroxyuracil in the transcribed strand, with hnRNP-U playing a critical role. Chromatin immunoprecipitation/reimmunoprecipitation studies showed association of NEIL2, RNA polymerase II, and hnRNP-U on transcribed but not on transcriptionally silent genes. Furthermore, NEIL2-depleted cells accumulated more DNA damage in active than in silent genes. These results strongly support the preferential role of NEIL2 in repairing oxidized bases in the transcribed genes of mammalian cells.
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