Background Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. Methods We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. Results We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. Conclusions This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.

The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu 2þ ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu 2þ coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu 2þ -ion based long-range distance constraints. Double electron-electron resonance data yield Cu 2þ -Cu 2þ and Cu 2þ -nitroxide distances that are uniquely consistent with one Cu 2þ bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu 2þ ions bind per complex. Unexpectedly, Mg 2þ -catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu 2þ binding at these hitherto unknown sites, 13 Å away from the Mg 2þ positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu 2þ ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu 2þ , the Mg 2þ -dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable doublestrand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg 2þ in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg 2þ . Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.T he biochemical basis of specificity in the interaction of proteins with DNA sites is a major problem of modern molecular genetics. Studies of many protein-DNA complexes by crystallography have elucidated the intermolecular recognition contacts (1), but it is clear that point-to-point contacts cannot fully explain specificity. Solution biochemical and computational studies have shown that a comprehensive view of specificity determination must also include factors such as shape recognition (2), mutual accommodation of the macromolecules through DNA distortion or conformational selection (1, 3-6), and/or DNA-induced protein folding (5, 7). Thermodynamic studies have revealed that specific protein-DNA association is often driven primarily by the favorable entropy increase provided by desolvation of the apposed complementary surfaces (8-10).For those DNA-binding proteins that are also DNA-modifying enzymes (nucleases, methylases, recombinases, repair enzymes, etc.) a key question is the relationship between DNA-binding specificity and catalytic specificity. One exemplary system for addressing specificity determination is the EcoRI endonuclease (3, 11), a 62 kDa homodimer that recognizes the DNA site 5′-GAATTC-3′ and binds as much as 90,000-fold better (3, 12) than a...

Kufor-Rakeb syndrome (KRS) is a rare form of autosomal recessive juvenile or early-onset, levodopa responsive parkinsonism and has been associated with mutations in ATP13A2(also known as PARK9), a lysosomal type 5 P-type ATPase. Recently, we identified novel compound heterozygous mutations, c.3176T>G (p.L1059R) and c.3253delC (p.L1085WfsX1088) in ATP13A2 of two siblings affected with KRS. When overexpressed, wild-type ATP13A2 localized to Lysotracker-positive and LAMP2-positive lysosomes while both truncating and missense mutated ATP13A2 were retained in the endoplasmic reticulum (ER). Both mutant proteins were degraded by the proteasomal but not the lysosomal pathways. In addition, ATP13A2 mRNA with c.3253delC was degraded by nonsense-mediated mRNA decay (NMD), which was protected by cycloheximide treatment. To validate our findings in a biologically relevant setting, we used patient-derived human olfactory neurosphere cultures and fibroblasts and demonstrated persistent ER stress by detecting upregulation of unfolded protein response-related genes in the patient-derived cells. We also confirmed NMD degraded ATP13A2 c.3253delC mRNA in the cells. These findings indicate that these novel ATP13A2 mutations are indeed pathogenic and support the notion that mislocalization of the mutant ATP13A2, resultant ER stress, alterations in the proteasomal pathways and premature degradation of mutant ATP13A2 mRNA contribute to the aetiology of KRS.

SynopsisThe ceric ion-cellulose redox system has been studied for grafting acrylonitrile on cotton fibers. Grafting yields are very high as compared to the persu1fat)ethiosulfate redox system reported earlier. Traces of copper sulfate in the reaction mixture do not increase grafting yields, unlike the persulfate-thiosulfate system. The high polymerization rate on cotton fibers is shown to be due to the reducing action of cellulose and not to the large surface area of cotton fibers. The Ce+4 consumption during grafting is higher than during oxidation of cellulose, indicating formation of homopolymer during the grafting reaction. Studies on the consumption of Ce+4 by model compounds siich as D-glucose and a-methyl-D-glucoside show that the hemiacetal group in D-glucose is responsible for a faster rate of Ce+' Consumption. Formation of a Ce+'-alcohol complex also contributes to the initial fast rate of Cef4 consumption. Studies on the oxidation of celldose by Ce+4 indicate that the initial oxidative attack occurs on carbon atom 2, with the formation of a >C=O group. On further oxidation, cleavage of the C2-G bond occurs as shown by the presence of glycol aldehyde determined chromatographically. Cellulose-polyacrylonitrile grafts have been isolated by an acetolysis treatment followed by extraction with dimethylformamide. Number-average molecular weight,s of the isolated fractions are approximately 50,000-55,000. A theoretical method to calculate the number-average molecular weights, based on the PAN and the COOH contents of the grafted cellulose, is described.