Age-related neurodegenerative disorders including Alzheimer's disease and Huntington's disease (HD) consistently show elevated DNA damage, but the relevant molecular pathways in disease pathogenesis remain unclear. One attractive gene is that encoding the ataxia-telangiectasia mutated (ATM) protein, a kinase involved in the DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been demonstrated in Alzheimer's disease and in mouse models of other neurodegenerative diseases. We show that ATM signaling was consistently elevated in cells derived from HD mice and in brain tissue from HD mice and patients. ATM knockdown protected from toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we show that genetic reduction of Atm gene dosage by one copy ameliorated multiple behavioral deficits and partially improved neuropathology. Small-molecule ATM inhibitors reduced mHTT-induced death of rat striatal neurons and induced pluripotent stem cells derived from HD patients. Our study provides converging genetic and pharmacological evidence that reduction of ATM signaling could ameliorate mHTT toxicity in cellular and animal models of HD, suggesting that ATM may be a useful therapeutic target for HD.

Polyglutamine diseases are characterized by neuronal intranuclear inclusions (NIIs) of expanded polyglutamine proteins, indicating the failure of protein degradation. UBB(+1), an aberrant form of ubiquitin, is a substrate and inhibitor of the proteasome, and was previously reported to accumulate in Alzheimer disease and other tauopathies. Here, we show accumulation of UBB(+1) in the NIIs and the cytoplasm of neurons in Huntington disease and spinocerebellar ataxia type-3, indicating inhibition of the proteasome by polyglutamine proteins in human brain. We found that UBB(+1) not only increased aggregate formation of expanded polyglutamines in neuronally differentiated cell lines, but also had a synergistic effect on apoptotic cell death due to expanded polyglutamine proteins. These findings implicate UBB(+1) as an aggravating factor in polyglutamine-induced neurodegeneration, and clearly identify an important role for the ubiquitin-proteasome system in polyglutamine diseases.

The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA. NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions. Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens. In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3 gene at nucleotide resolution. In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage. Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate. Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA. The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcriptioncoupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context. UV light induces two major classes of genotoxic lesions in DNA, i.e., cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4)-pyrimidone photoproducts (6-4PPs). Both lesions are repaired by the nucleotide excision repair (NER) pathway, in which incision of the damaged strand on both sides of the lesion is followed by resynthesis of excised DNA with the undamaged strand as a template (reviewed in reference 2). Although the molecular mechanism of the NER reaction has become increasingly clear as it has been reconstituted in vitro by using purified components of either yeast or human origin (1,4,15), the mechanism of damage recognition in the nucleus where DNA is folded into chromatin with different levels of complexity is largely unknown. One possible way by which cells sense DNA damage is lesion interference with essential cellular DNA metabolic processes, like transcription, replication, or even recombination. This is exemplified by the intimate link found for the process of NER and mRNA transcription: UVinduced CPDs introduced in sequences transcribed by RNA polymerase or RNA polymerase II, respectively, in pro-and eukaryotes, are repaired preferentially to CPDs induced in nontranscribed DNA (13,14). The molecular basis for this enhanced strand-specific repair, more commonly termed transcription-coupled repair (TCR) since it is dependent upon ongoing transcription (10, 18), is thought to originate from efficient recruitment of repair proteins to...

No abstract

Genes positioned close to telomeres in yeast are silenced by a heterochromatin-like structure containing Sir proteins. To investigate whether silencing also affects DNA repair, we studied removal of UV lesions by photolyase and nucleotide excision repair (NER) in strains containing the URA3 gene inserted 2 kilobases from a telomere. URA3 was transcriptionally active in sir3⌬ mutants, partially silenced in SIR3 cells, or completely silenced by overexpression of SIR3 or deletion of RPD3. The active URA3 showed efficient repair by both pathways. Fast repair of the promoter and 3 end by photolyase reflected a non-nucleosomal structure. Partial silencing had no remarkable effect on photolyase but reduced repair by NER, indicating differential accessibility for the two repair reactions. Complete silencing inhibits NER and photolyase in the coding region as well as in the promoter and the 3 -end. Conventional nuclease footprinting analyses revealed subtle changes in the promoter proximal nucleosome under partially silenced conditions but a pronounced reorganization of chromatin extending over the whole gene in silenced chromatin. Thus, both repair systems are sensitive to chromatin changes associated with silencing and provide direct evidence for a compact structure of heterochromatin.Silencing refers to transcriptional inhibition characterized by the epigenetic formation of a repressive chromatin structure, frequently referred to as heterochromatin. In yeast Saccharomyces cerevisiae, silencing occurs at several genetic loci, including the cryptic mating-type loci (HML and HMR), the ribosomal DNA, and regions close to telomeres (1-4). Silencing of genes integrated in subtelomeric regions decreases with increasing distance from the telomere (telomere position effect) (5). In contrast to stable silencing at the HM loci, silencing of subtelomeric genes is variegated, resulting in stochastic patterns of repression of transcription (6 -9).Telomeric silencing depends on numerous proteins: three silent information regulators (Sir2, Sir3, and Sir4), histones, proteins required for chromatin assembly, proteins involved in telomere formation as well as enzymes that modify histones by deacetylation, ubiquitination, and methylation (5-7, 10 -16). Silencing is enforced by the proximity to a pool of concentrated Sir proteins, clustered telomeres, and perinuclear localization (17)(18)(19)(20). Perinuclear localization appears to be necessary, albeit not sufficient, for silencing (21). Current models propose a stepwise formation of telomeric heterochromatin (2, 4, 22). Rap1 binds to telomeric Rap1-binding sites and recruits Sir4. Sir4 recruits Sir3 and the NAD-dependent histone deacetylase Sir2. Sir2 deacetylates histone H4 at Lys-16, which allows Sir3 binding to nucleosomes. Sir3 recruits more Sir4 onto nucleosomes, and the process is repeated as the SIR complex spreads along chromatin away from the initiation site (23, 24). In addition, telomeric chromatin appears to be further stabilized by folding back of the telomere, which allow...