SummaryALC1, a novel PARP1-stimulated chromatin-remodelling enzyme promotes DNA repair.Post-translational modifications play key roles in orchestrating chromatin plasticity. Although various chromatin-remodelling enzymes have been described that respond to specific histone modifications, little is known about the role of poly(ADP-ribose) in chromatin remodelling. Here, we identify a novel chromatin-remodelling enzyme, ALC1 (Amplified in Liver Cancer 1), that is specifically regulated by poly(ADP-ribosyl) ation. ALC1 binds poly(ADP-ribose) via a C-terminal Macro domain and catalyzes PARP1-stimulated nucleosome sliding, conferred by an N-terminal ISWI-related helicase core. Our results define ALC1 as a novel DNA damage-response protein, whose role in this process is sustained by its association with known DNA repair factors and its rapid poly(ADP-ribose)-dependent recruitment to DNA damage sites. Furthermore, we show that depletion or overexpression of ALC1 results in sensitivity to DNA-damaging agents. Collectively, these results provide new insights into the mechanisms by which poly(ADP-ribose) regulates DNA repair.The restricted accessibility of DNA within chromatin presents a barrier to DNA manipulations that require direct protein-DNA interactions (1-3). Processes such as transcription, repair and replication that require efficient DNA recognition are therefore dependent on the appropriate modulation of chromatin structure. Chromatin relaxation is a critical event that occurs during DNA repair and is associated with post-translational poly(ADP-ribose) (PAR) modification (4). PAR is synthesized in a reaction that utilizes NAD+ as a substrate by the PARP family of enzymes, of which PARP1 (and to a lesser extent PARP2) respond to DNA strand breaks (5-7). As a consequence of poly(ADP-

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.

Post-translational modification (PTM) of proteins plays an important part in mediating protein interactions and/or the recruitment of specific protein targets. PTM can be mediated by the addition of functional groups (for example, acetylation or phosphorylation), peptides (for example, ubiquitylation or sumoylation), or nucleotides (for example, poly(ADP-ribosyl)ation). Poly(ADP-ribosyl)ation often involves the addition of long chains of ADP-ribose units, linked by glycosidic ribose-ribose bonds, and is critical for a wide range of processes, including DNA repair, regulation of chromosome structure, transcriptional regulation, mitosis and apoptosis. Here we identify a novel poly(ADP-ribose)-binding zinc finger (PBZ) motif in a number of eukaryotic proteins involved in the DNA damage response and checkpoint regulation. The PBZ motif is also required for post-translational poly(ADP-ribosyl)ation. We demonstrate interaction of poly(ADP-ribose) with this motif in two representative human proteins, APLF (aprataxin PNK-like factor) and CHFR (checkpoint protein with FHA and RING domains), and show that the actions of CHFR in the antephase checkpoint are abrogated by mutations in PBZ or by inhibition of poly(ADP-ribose) synthesis.

The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells 1 , 2 . Upon binding to genomic lesions, these enzymes utilise NAD + to modify a plethora of proteins with mono- and poly(ADP-ribose) signals that are important for subsequent chromatin decompaction and repair factor recruitment 3 , 4 . These post-translational modification events are predominantly serine-linked and require HPF1, an accessory factor that is specific for DNA damage response and switches the amino-acid specificity of PARP1/2 from aspartate/glutamate to serine residues 5 – 10 . Here, we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from both PARP1/2 and HPF1. We further show that the assembly of this new catalytic centre is essential for DNA damage-induced protein ADP-ribosylation in human cells. In response to DNA damage and NAD + binding site occupancy, the HPF1-PARP1/2 interaction is enhanced via allosteric networks operating within PARP1/2, providing an additional level of regulation in DNA repair induction. As HPF1 forms a joint active site with PARP1/2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.

Methanogenic archaea possess unusual seryl-tRNA synthetase (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. The two types of SerRSs show only minimal sequence similarity, primarily within class II conserved motifs 1, 2 and 3. Here, we report a 2.5 Å resolution crystal structure of the atypical methanogenic Methanosarcina barkeri SerRS and its complexes with ATP, serine and the nonhydrolysable seryl-adenylate analogue 5 0 -O-(N-serylsulfamoyl)adenosine. The structures reveal two idiosyncratic features of methanogenic SerRSs: a novel N-terminal tRNA-binding domain and an active site zinc ion. The tetra-coordinated Zn 2 þ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461) and binds the amino group of the serine substrate. The absolute requirement of the metal ion for enzymatic activity was confirmed by mutational analysis of the direct zinc ion ligands. This zinc-dependent serine recognition mechanism differs fundamentally from the one employed by the bacterial-type SerRSs. Consequently, SerRS represents the only known aminoacyl-tRNA synthetase system that evolved two distinct mechanisms for the recognition of the same amino-acid substrate.