ABSTRACT
PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways.
Subject(s)
ADP-Ribosylation , Interferons , Poly(ADP-ribose) Polymerases , Ubiquitin-Protein Ligases , Humans , Poly(ADP-ribose) Polymerases/metabolism , Poly(ADP-ribose) Polymerases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Interferons/metabolism , Ubiquitination , HEK293 Cells , SARS-CoV-2/metabolism , Signal Transduction , COVID-19/virology , COVID-19/metabolism , Neoplasm ProteinsABSTRACT
Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.
Subject(s)
ADP-Ribosylation , Nucleic Acids , Ubiquitin-Protein Ligases , Adenosine Diphosphate Ribose/metabolism , Nucleic Acids/metabolism , Okadaic Acid/analogs & derivatives , Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , HumansABSTRACT
The timely removal of ADP-ribosylation is crucial for efficient DNA repair. However, much remains to be discovered about ADP-ribosylhydrolases. Here, we characterize the physiological role of TARG1, an ADP-ribosylhydrolase that removes aspartate/glutamate-linked ADP-ribosylation. We reveal its function in the DNA damage response and show that the loss of TARG1 sensitizes cells to inhibitors of topoisomerase II, ATR, and PARP. Furthermore, we find a PARP1-mediated synthetic lethal interaction between TARG1 and PARG, driven by the toxic accumulation of ADP-ribosylation, that induces replication stress and genomic instability. Finally, we show that histone PARylation factor 1 (HPF1) deficiency exacerbates the toxicity and genomic instability induced by excessive ADP-ribosylation, suggesting a close crosstalk between components of the serine- and aspartate/glutamate-linked ADP-ribosylation pathways. Altogether, our data identify TARG1 as a potential biomarker for the response of cancer cells to PARP and PARG inhibition and establish that the interplay of TARG1 and PARG protects cells against genomic instability.
Subject(s)
Aspartic Acid , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Aspartic Acid/metabolism , ADP-Ribosylation , Genomic Instability , Glutamates/metabolism , Carrier Proteins/metabolism , Nuclear Proteins/metabolismABSTRACT
PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.
Subject(s)
COVID-19 , Transferases , Humans , Poly(ADP-ribose) Polymerase Inhibitors , Antiviral Agents , Hydrolases , Poly(ADP-ribose) Polymerases/geneticsABSTRACT
AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.
Subject(s)
Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases , Humans , Poly(ADP-ribose) Polymerases/metabolism , Protein Domains , ADP-Ribosylation , RNA/metabolismABSTRACT
Structurally complex genomic regions, such as centromeres, are inherently difficult to duplicate. The mechanism behind centromere inheritance is not well understood, and one of the key questions relates to the reassembly of centromeric chromatin following DNA replication. Here, we define ERCC6L2 as a key regulator of this process. ERCC6L2 accumulates at centromeres and promotes deposition of core centromeric factors. Interestingly, ERCC6L2-/- cells show unrestrained replication of centromeric DNA, likely caused by the erosion of centromeric chromatin. Beyond centromeres, ERCC6L2 facilitates replication at genomic repeats and non-canonical DNA structures. Notably, ERCC6L2 interacts with the DNA-clamp PCNA through an atypical peptide, presented here in a co-crystal structure. Finally, ERCC6L2 also restricts DNA end resection, acting independently of the 53BP1-REV7-Shieldin complex. We propose a mechanistic model, which reconciles seemingly distinct functions of ERCC6L2 in DNA repair and DNA replication. These findings provide a molecular context for studies linking ERCC6L2 to human disease.
Subject(s)
Centromere , Chromatin , Humans , Centromere/metabolism , DNA/chemistry , DNA Replication , DNA Repair , DNA Helicases/metabolismABSTRACT
Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged. Here, we show that DELTEX E3 ligases specifically target the 3' hydroxyl of the adenosine diphosphate (ADP)-ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3~ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2~ubiquitin onto a substrate. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad-like manner. In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells.
ABSTRACT
Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.
Subject(s)
Adenosine Diphosphate/metabolism , Carrier Proteins/metabolism , Glycoside Hydrolases/metabolism , Nuclear Proteins/metabolism , ADP-Ribosylation , Carrier Proteins/genetics , Cell Line, Tumor , DNA Damage , Gene Knockout Techniques , Glycoside Hydrolases/genetics , Histones/metabolism , Humans , Nuclear Proteins/genetics , Protein Processing, Post-Translational , Proteome/metabolism , Proteomics , Serine/metabolismABSTRACT
Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.
Subject(s)
Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Poly ADP Ribosylation/physiology , ADP-Ribosylation , Adenosine Diphosphate Ribose/metabolism , Animals , Catalysis , Humans , Hydrolysis , Poly ADP Ribosylation/genetics , Protein Processing, Post-Translational , Wnt Signaling PathwayABSTRACT
Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.
Subject(s)
Carrier Proteins/metabolism , DNA Damage , DNA Repair , Neoplasms/drug therapy , Nuclear Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Serine/metabolism , ADP-Ribosylation , Cell Line , Cell Line, Tumor , Humans , Neoplasms/enzymology , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Protein Processing, Post-TranslationalABSTRACT
ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.
Subject(s)
Glycoside Hydrolases/metabolism , Poly ADP Ribosylation/physiology , ADP-Ribosylation , Adenosine Diphosphate Ribose/metabolism , Cell Line, Tumor , Chromatin , DNA , DNA Damage , Fibroblasts/metabolism , Glycoside Hydrolases/genetics , Glycoside Hydrolases/physiology , HEK293 Cells , HeLa Cells , Humans , Poly Adenosine Diphosphate Ribose/metabolism , Primary Cell CultureABSTRACT
Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.
Subject(s)
Chromatin Assembly and Disassembly , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Neoplasm Proteins/metabolism , Neoplasms, Experimental/metabolism , Nucleosomes/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , DNA Helicases/genetics , DNA Replication/drug effects , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-Binding Proteins/genetics , Homologous Recombination/drug effects , Mice , Mice, Knockout , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasms, Experimental/genetics , Nucleosomes/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/geneticsABSTRACT
Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.
Subject(s)
ADP-Ribosylation , Molecular Docking Simulation , Poly(ADP-ribose) Polymerases/chemistry , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Diphosphate Ribose/chemistry , Adenosine Diphosphate Ribose/metabolism , Binding Sites , Evolution, Molecular , Humans , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Domains , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. After binding to genomic lesions, these enzymes use NAD+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues5-10. 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 HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.
Subject(s)
ADP-Ribosylation , Carrier Proteins/chemistry , Carrier Proteins/metabolism , DNA Damage , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Allosteric Regulation , Amino Acid Motifs , Amino Acid Sequence , Animals , Biocatalysis , Carrier Proteins/genetics , Catalytic Domain , HEK293 Cells , Humans , Models, Molecular , Mutation , NAD/metabolism , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Sea AnemonesABSTRACT
Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations.
Subject(s)
DNA Helicases/chemistry , DNA Helicases/metabolism , DNA Replication , Adenosine Triphosphate/metabolism , Amino Acid Motifs , DNA Helicases/genetics , Genomic Instability , Humans , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein DomainsABSTRACT
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(ADP-ribose) (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.
Subject(s)
Glycoside Hydrolases/physiology , Neurodegenerative Diseases/enzymology , Poly Adenosine Diphosphate Ribose/physiology , Thiolester Hydrolases/physiology , Amino Acid Sequence , Base Sequence , Cells, Cultured , Child , Child, Preschool , Family , Female , Glycoside Hydrolases/genetics , HEK293 Cells , HeLa Cells , Humans , Male , Models, Molecular , Molecular Sequence Data , Neurodegenerative Diseases/genetics , Pedigree , Poly Adenosine Diphosphate Ribose/genetics , Protein Processing, Post-Translational/genetics , Sequence Homology, Amino Acid , Thiolester Hydrolases/geneticsABSTRACT
To efficiently duplicate their genomic content, cells must overcome DNA lesions that interfere with processive DNA replication. These lesions may be removed and repaired, rather than just tolerated, to allow continuity of DNA replication on an undamaged DNA template. However, it is unclear how this is achieved at a molecular level. Here we identify a new replication-associated factor, ZRANB3 (zinc finger, RAN-binding domain containing 3), and propose its role in the repair of replication-blocking lesions. ZRANB3 has a unique structure-specific endonuclease activity, which is coupled to ATP hydrolysis. It cleaves branched DNA structures with unusual polarity, generating an accessible 3'-OH group in the template of the leading strand. Furthermore, ZRANB3 localizes to DNA replication sites and interacts with the components of the replication machinery. It is recruited to damaged replication forks via multiple mechanisms, which involve interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures. Collectively, our data support a role for ZRANB3 in the replication stress response and suggest new insights into how DNA repair is coordinated with DNA replication to maintain genome stability.
Subject(s)
DNA Helicases/metabolism , DNA Repair/physiology , DNA Replication/physiology , Endonucleases/metabolism , Genomic Instability/physiology , Stress, Physiological/physiology , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , DNA/biosynthesis , DNA/genetics , DNA Helicases/genetics , Endonucleases/genetics , HeLa Cells , Humans , Hydrolysis , Polyubiquitin/genetics , Polyubiquitin/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Substrate Specificity/physiologyABSTRACT
Poly(ADP-ribosyl)ation plays a major role in DNA repair, where it regulates chromatin relaxation as one of the critical events in the repair process. However, the molecular mechanism by which poly(ADP-ribose) modulates chromatin remains poorly understood. Here we identify the poly(ADP-ribose)-regulated protein APLF as a DNA-damage-specific histone chaperone. APLF preferentially binds to the histone H3/H4 tetramer via its C-terminal acidic motif, which is homologous to the motif conserved in the histone chaperones of the NAP1L family (NAP1L motif). We further demonstrate that APLF exhibits histone chaperone activities in a manner that is dependent on its acidic domain and that the NAP1L motif is critical for the repair capacity of APLF in vivo. Finally, we identify structural analogs of APLF in lower eukaryotes with the ability to bind histones and localize to the sites of DNA-damage-induced poly(ADP-ribosyl)ation. Collectively, these findings define the involvement of histone chaperones in poly(ADP-ribose)-regulated DNA repair reactions.
Subject(s)
DNA Repair , Histone Chaperones/physiology , Phosphoproteins/physiology , Amino Acid Motifs , Animals , Caenorhabditis elegans/genetics , Cell Line , DNA Damage , DNA-(Apurinic or Apyrimidinic Site) Lyase , Eukaryota/genetics , HeLa Cells , Histone Chaperones/chemistry , Histone Chaperones/genetics , Histones/metabolism , Humans , Molecular Sequence Data , Phosphoproteins/chemistry , Phosphoproteins/genetics , Poly-ADP-Ribose Binding Proteins , Protein Interaction Mapping , Proteins/chemistry , Proteins/genetics , Proteins/physiology , Sequence Homology , tRNA MethyltransferasesABSTRACT
Posttranslational modifications play key roles in regulating chromatin plasticity. Although various chromatin-remodeling enzymes have been described that respond to specific histone modifications, little is known about the role of poly[adenosine 5'-diphosphate (ADP)-ribose] in chromatin remodeling. Here, we identify a chromatin-remodeling enzyme, ALC1 (Amplified in Liver Cancer 1, also known as CHD1L), that interacts with poly(ADP-ribose) and catalyzes PARP1-stimulated nucleosome sliding. Our results define ALC1 as a 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.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , DNA Helicases/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Cell Line , DNA Damage , DNA Helicases/chemistry , DNA Helicases/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Humans , Hydrogen Peroxide/pharmacology , Immunoprecipitation , Kinetics , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Nucleosomes/metabolism , Phleomycins/pharmacology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Protein Structure, Tertiary , Radiation, Ionizing , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
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.