Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADPribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg 2+ resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here we present the first crystal structure of a pre-catalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg 2+. This catalytic intermediate was trapped with a non-hydrolyzable deoxynucleotide analogue. Comparison with two new structures of DNA polymerase β lacking the 3'-OH or catalytic Mg 2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the αP of the incoming nucleotide.

The structure of epothilone A, bound to alpha,beta-tubulin in zinc-stabilized sheets, was determined by a combination of electron crystallography at 2.89 angstrom resolution and nuclear magnetic resonance-based conformational analysis. The complex explains both the broad-based epothilone structure-activity relationship and the known mutational resistance profile. Comparison with Taxol shows that the longstanding expectation of a common pharmacophore is not met, because each ligand exploits the tubulin-binding pocket in a unique and independent manner.