Spinocerebellar ataxia type 3 (SCA3), aka Machado-Joseph Disease is the most common dominantly inherited ataxia worldwide. It is the result of a CAG (glutamine) repeat expansion in the coding region of Ataxin 3, a polyglutamine (14-41 repeats)-containing protein. While investigating the mechanism of SCA3, we have found that wild type Ataxin 3 stimulates, while the mutant form abrogates the activity of polynucleotide kinase 3?- phosphatase (PNKP), an essential DNA repair protein. This resulted in the accumulation of DNA double-strand breaks in the brains of SCA3 patients and mice. Constant activation of the DNA damage-response pathway with consequent cellular apoptosis is a plausible cause of SCA3. Our recent studies have revealed that PNKP plays a critical role in DNA double strand break repair via classical non-homologous end joining (C-NHEJ). We have demonstrated that PNKP-mediated C-NHEJ repair pathway is error-free, with homologous nascent RNA providing the template to restore the missing sequence at the double strand break site in transcribed genes. DNA strand break analysis in the SCA3 mouse brain versus wild type showed significantly more strand breaks in the transcribed but, not the non-transcribed genes. Collectively, these data indicate that differential genomic region-specific strand break repair occurs in neuronal cells. Our lab found that, in addition to ATXN3, two RNA- binding/splicing proteins (NONO and SFPQ) and a key allosteric regulator of glycolysis are involved in this pathway. We are the first to show the roles of Ataxin 3 and the glycolysis-regulatory protein in classical non- homologous end joining mediated DNA double strand break repair. Notably, all these factors form a physiological complex with RNA polymerase II and other classical non-homologous end joining proteins. We also found that NONO and SFPQ significantly stimulated PNKP?s end-processing activity. We postulate that these RNA-binding proteins facilitate the formation of the RNA-DNA hybrid so that the RNA-dependent DNA polymerase can effectively use the RNA as a template to restore the missing information. However, the glycolysis inducer did not affect PNKP?s activity, but the inducer-catalyzed metabolite significantly stimulated PNKP?s activity. Furthermore, the metabolite can even restore the activity of PNKP in SCA3 patients? brain nuclear extract, suggesting the promising therapeutic potential of this natural metabolite for SCA3. Hence, this project will test the hypotheses that: restoration of the PNKP-mediated C-NHEJ repair pathway is crucial for maintaining the integrity of the transcribed genome, and thereby rescuing neuronal cells from the deleterious effects of mutant ATXN3. Understanding the mechanistic basis of error-free double strand break repair of the transcribed genes in neuronal cells via classical non-homologous end joining pathway, and the modulatory effect of a natural metabolite in such a pathway, will significantly advance our knowledge and should accelerate the development of new treatment modalities for SCA3 and other polyQ diseases.

Public Health Relevance

Our studies are designed to establish the mechanistic basis for error-free repair of DNA double-strand breaks in the transcribed genome. Defects in this process in neuronal cells could play a causal role in the development of various human neurological disorders. Understanding the molecular mechanisms underlying this DNA repair pathway should facilitate the development of novel therapeutic strategies to prevent the onset of or treat human diseases that are causally linked to genomic instability.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS073976-06A1
Application #
9605560
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Miller, Daniel L
Project Start
2012-07-01
Project End
2022-04-30
Budget Start
2018-07-15
Budget End
2019-04-30
Support Year
6
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Texas Med Br Galveston
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
Sahan, Ayse Z; Hazra, Tapas K; Das, Soumita (2018) The Pivotal Role of DNA Repair in Infection Mediated-Inflammation and Cancer. Front Microbiol 9:663
Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana et al. (2016) Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes. Nat Commun 7:13049
Chatterjee, Arpita; Saha, Saikat; Chakraborty, Anirban et al. (2015) The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis. PLoS Genet 11:e1004749
Mandal, Santi M; Chakraborty, Anirban; Hossain, Maidul et al. (2015) Amphotericin B and anidulafungin directly interact with DNA and induce oxidative damage in the mammalian genome. Mol Biosyst 11:2551-9
Suganya, Rangaswamy; Chakraborty, Anirban; Miriyala, Sumitra et al. (2015) Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease. DNA Repair (Amst) 27:40-8
Chakraborty, Anirban; Wakamiya, Maki; Venkova-Canova, Tatiana et al. (2015) Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation. J Biol Chem 290:24636-48
Gao, Rui; Liu, Yongping; Silva-Fernandes, Anabela et al. (2015) Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3. PLoS Genet 11:e1004834
Hegde, Pavana M; Dutta, Arijit; Sengupta, Shiladitya et al. (2015) The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD. J Biol Chem 290:20919-33
Hajas, Gyorgy; Bacsi, Attila; Aguilera-Aguirre, Leopoldo et al. (2013) 8-Oxoguanine DNA glycosylase-1 links DNA repair to cellular signaling via the activation of the small GTPase Rac1. Free Radic Biol Med 61:384-94
Hegde, Muralidhar L; Hegde, Pavana M; Bellot, Larry J et al. (2013) Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins. Proc Natl Acad Sci U S A 110:E3090-9

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