Our work focuses on two main project areas, i) DNA damage recognition and processing, and ii) DNA damage signaling. Progress in the last year in these areas is summarized below: i) DNA damage recognition and processing. Aptx is a conserved eukaryotic DNA repair enzyme that is important for protection of cells from oxidative DNA damage, and APTX mutations cause the hereditary neurodegenerative disorder Ataxia with Oculomotor Apraxia 1 (AOA1). In the ultimate step of DNA replication and repair processes, DNA ligases seal DNA nicks through with a mechanism that can abort when the ligase encounters damaged DNA. Such "abortive ligation" generates a secondary form of damage, 5'-adenylated DNA-termini, which is corrected by Aptx to protect genomic integrity. To understand the context for Aprataxin (Aptx) deadenylation repair we examined the importance of Aptx to RNaseH2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We demonstrated that DNA ligases generate adenylated 5′-ends containing a ribose characteristic of RNaseH2 incision. Aptx efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human Aptx/RNA-DNA/AMP/Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA-binding, proper protein folding and conformational changes, all of which are impacted by heritable APTX mutations in Ataxia with Oculomotor Apraxia 1 (AOA1). Together, these results suggest that accumulation of adenylated RNA-DNA may contribute to neurological disease. ii) DNA damage signaling. ADP-ribosylation is a reversible post-translational protein modification implicated in a range of cellular processes, including DNA repair, transcriptional regulation, cell differentiation and proliferation, inflammatory and immune responses, and apoptosis. PARPs use NAD+ as a substrate and covalently attach an ADP-ribose nucleotide, predominantly to the carboxyl group of glutamate residues on target proteins. Some PARP family members can subsequently add additional ADP-ribose units through glycosidic ribose-ribose bonds to generate a PAR chain specific hydrolysis of ribose-ribose bonds in PAR chains is catalysed by PAR glycohydrolase (PARG), but PARG is unable to cleave the ester bond between the ADP-ribose unit and the glutamate. We identified and structurally characterized an enzymatic activity in the human macrodomain containing protein C6orf130 that catalyses this step of PAR catabolism. We propose a cellular role for C6orf130 protein in the removal of the terminal ADP-ribose unit linked to PARP-modified proteins, by directly reversing protein mono(ADP-ribosyl)ation or by completing the reversal of protein poly(ADP-ribosyl)ation following the PARG reaction. Hence we have renamed this protein Terminal ADP-Ribose protein Glycohydrolase (TARG1). X-ray structures of C6orf130/TARG1 and biochemical data suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130/TARG1 protein from cells leads to proliferation and DNA repair defects, and homozygous mutation of the C6orf130 gene is found in patients with severe neurodegeneration. In ongoing studies, we are testing hypotheses that TARG1 activity plays roles in: 1) Catabolism of PAR chains in conjunction with PARG (poly-adp-ribose glycohydrolase) or, 2) Down-regulation of Parp1 signaling through direct reversal (removal of mono-ADP-ribose) of Parp1 and ADPR-modified proteins.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2013
Total Cost
$1,642,735
Indirect Cost
City
State
Country
Zip Code
Andres, Sara N; Schellenberg, Matthew J; Wallace, Bret D et al. (2015) Recognition and repair of chemically heterogeneous structures at DNA ends. Environ Mol Mutagen 56:21-Jan
Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver et al. (2014) ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling. EMBO J 33:482-500
Gao, Rui; Schellenberg, Matthew J; Huang, Shar-Yin N et al. (2014) Proteolytic degradation of topoisomerase II (Top2) enables the processing of Top2┬ĚDNA and Top2┬ĚRNA covalent complexes by tyrosyl-DNA-phosphodiesterase 2 (TDP2). J Biol Chem 289:17960-9
Tumbale, Percy; Williams, Jessica S; Schellenberg, Matthew J et al. (2014) Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity. Nature 506:111-5
Sharifi, Reza; Morra, Rosa; Appel, C Denise et al. (2013) Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease. EMBO J 32:1225-37
Crown, K Nicole; Savytskyy, Oleksandr P; Malik, Shehre-Banoo et al. (2013) A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation. G3 (Bethesda) 3:1927-43
Adhikari, Sanjay; Karmahapatra, Soumendra K; Elias, Hadi et al. (2011) Development of a novel assay for human tyrosyl DNA phosphodiesterase 2. Anal Biochem 416:112-6
Schellenberg, Matthew J; Williams, R Scott (2011) DNA end processing by polynucleotide kinase/phosphatase. Proc Natl Acad Sci U S A 108:20855-6
Williams, Gareth J; Williams, R Scott; Williams, Jessica S et al. (2011) ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair. Nat Struct Mol Biol 18:423-31
Williams, R Scott; Kunkel, Thomas A (2011) FEN nucleases: bind, bend, fray, cut. Cell 145:171-2