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Nature Cell Biology Apr 2022DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA...
DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA polymerase II (Pol II) is not fully understood. Central to the DNA damage response, poly(ADP-ribose) polymerase 1 (PARP1) initiates DNA repair by translocating to the lesions where it catalyses protein poly(ADP-ribosylation). Here we report that PARP1 inhibits Pol II elongation by inactivating the transcription elongation factor P-TEFb, a CDK9-cyclin T1 (CycT1) heterodimer. After sensing damage, the activated PARP1 binds to transcriptionally engaged P-TEFb and modifies CycT1 at multiple positions, including histidine residues that are rarely used as an acceptor site. This prevents CycT1 from undergoing liquid-liquid phase separation that is required for CDK9 to hyperphosphorylate Pol II and to stimulate elongation. Functionally, poly(ADP-ribosylation) of CycT1 promotes DNA repair and cell survival. Thus, the P-TEFb-PARP1 signalling plays a protective role in transcription quality control and genomic stability maintenance after DNA damage.
Topics: ADP-Ribosylation; Cyclin T; DNA Damage; Positive Transcriptional Elongation Factor B; RNA Polymerase II
PubMed: 35393539
DOI: 10.1038/s41556-022-00872-5 -
DNA Repair Sep 2021ADP-ribosylation is a chemical modification of macromolecules found across all domains of life and known to regulate a variety of cellular processes. Notably, it has a... (Review)
Review
ADP-ribosylation is a chemical modification of macromolecules found across all domains of life and known to regulate a variety of cellular processes. Notably, it has a well-established role in the DNA damage response. While it was historically known as a post-translational modification of proteins, recent studies have shown that nucleic acids can also serve as substrates of reversible ADP-ribosylation. More precisely, ADP-ribosylation of DNA bases, phosphorylated DNA ends and phosphorylated RNA ends have been reported. We will discuss these three types of modification in details. In a variety of bacterial species, including Mycobacterium tuberculosis, ADP-ribosylation of thymidine has emerged as the mode of action of a toxin-antitoxin system named DarTG, with the resultant products perceived as DNA damage by the cell. On the other hand, mammalian DNA damage sensors PARP1, PARP2 and PARP3 were shown to ADP-ribosylate phosphorylated ends of double-stranded DNA in vitro. Additionally, TRPT1 and several PARP enzymes, including PARP10, can add ADP-ribose to the 5'-phosphorylated end of single-stranded RNA in vitro, representing a novel RNA capping mechanism. Together, these discoveries have led to the emergence of a new and exciting research area, namely DNA and RNA ADP-ribosylation, that is likely to have far-reaching implications for the fields of DNA repair, replication and epigenetics.
Topics: ADP-Ribosylation; Animals; Cell Cycle Proteins; DNA; DNA Damage; DNA Repair; Humans; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; RNA
PubMed: 34116477
DOI: 10.1016/j.dnarep.2021.103144 -
ELife Aug 2017The modification of serines by molecules of ADP-ribose plays an important role in signaling that the DNA in a cell has been damaged and needs to be repaired.
The modification of serines by molecules of ADP-ribose plays an important role in signaling that the DNA in a cell has been damaged and needs to be repaired.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Hydrolases; Serine; Signal Transduction
PubMed: 28796599
DOI: 10.7554/eLife.29942 -
Molecular Cell May 2023PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a...
PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.
Topics: Histones; Poly (ADP-Ribose) Polymerase-1; ADP-Ribosylation; Chromatin; DNA Damage; Antibodies; Signal Transduction
PubMed: 37116497
DOI: 10.1016/j.molcel.2023.03.027 -
Molecular Cell Jul 2022The efficiency of homologous recombination (HR) in the repair of DNA double-strand breaks (DSBs) is closely associated with genome stability and tumor response to...
The efficiency of homologous recombination (HR) in the repair of DNA double-strand breaks (DSBs) is closely associated with genome stability and tumor response to chemotherapy. While many factors have been functionally characterized in HR, such as TOPBP1, their precise regulation remains unclear. Here, we report that TOPBP1 interacts with the RNA-binding protein HTATSF1 in a cell-cycle- and phosphorylation-dependent manner. Mechanistically, CK2 phosphorylates HTATSF1 to facilitate binding to TOPBP1, which promotes S-phase-specific TOPBP1 recruitment to damaged chromatin and subsequent RPA/RAD51-dependent HR, genome integrity, and cancer-cell viability. The localization of HTATSF1-TOPBP1 to DSBs is potentially independent of the transcription-coupled RNA-binding and processing capacity of HTATSF1 but rather relies on the recognition of poly(ADP-ribosyl)ated RPA by HTATSF1, which can be blunted with PARP inhibitors. Together, our study provides a mechanistic insight into TOPBP1 loading at HR-prone DSB sites via HTATSF1 and reveals how RPA-RAD51 exchange is tuned by a PARylation-phosphorylation cascade.
Topics: DNA Breaks, Double-Stranded; DNA Repair; Homologous Recombination; Phosphorylation; Poly ADP Ribosylation; Rad51 Recombinase
PubMed: 35597237
DOI: 10.1016/j.molcel.2022.04.031 -
Nature Communications Jun 2023In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors....
In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.
Topics: Animals; Serine; Drosophila; Drosophila melanogaster; ADP-Ribosylation; Poly Adenosine Diphosphate Ribose; Adenosine Diphosphate Ribose; Mammals
PubMed: 37268618
DOI: 10.1038/s41467-023-38793-y -
Molecular Cell Apr 2022Synthetic lethality through combinatorial targeting DNA damage response (DDR) pathways provides exciting anticancer therapeutic benefit. Currently, the long noncoding...
Synthetic lethality through combinatorial targeting DNA damage response (DDR) pathways provides exciting anticancer therapeutic benefit. Currently, the long noncoding RNAs (lncRNAs) have been implicated in tumor drug resistance; however, their potential significance in DDR is still largely unknown. Here, we report that a human lncRNA, CTD-2256P15.2, encodes a micropeptide, named PAR-amplifying and CtIP-maintaining micropeptide (PACMP), with a dual function to maintain CtIP abundance and promote poly(ADP-ribosyl)ation. PACMP not only prevents CtIP from ubiquitination through inhibiting the CtIP-KLHL15 association but also directly binds DNA damage-induced poly(ADP-ribose) chains to enhance PARP1-dependent poly(ADP-ribosyl)ation. Targeting PACMP alone inhibits tumor growth by causing a synthetic lethal interaction between CtIP and PARP inhibitions and confers sensitivity to PARP/ATR/CDK4/6 inhibitors, ionizing radiation, epirubicin, and camptothecin. Our findings reveal that a lncRNA-derived micropeptide regulates cancer progression and drug resistance by modulating DDR, whose inhibition could be employed to augment the existing anticancer therapeutic strategies.
Topics: DNA Repair; Endodeoxyribonucleases; Humans; Microfilament Proteins; Neoplasms; Peptides; Poly ADP Ribosylation; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; RNA, Long Noncoding
PubMed: 35219381
DOI: 10.1016/j.molcel.2022.01.020 -
Organic & Biomolecular Chemistry Jun 2019ADP-ribosylation is an important post-translational modification that plays a pivotal role in many cellular processes, including cell signaling, DNA repair, gene... (Review)
Review
ADP-ribosylation is an important post-translational modification that plays a pivotal role in many cellular processes, including cell signaling, DNA repair, gene regulation and apoptosis. Although chemical synthesis of mono- or poly-ADP-ribosylated biomolecules is extremely difficult due to the challenges in regio- and stereoselective glycosylation, suitable protective group manipulations and pyrophosphate construction, synthetic procedures towards these bio-related targets have been reported in recent years. Chemically synthesized well-defined ADP-ribose derivatives serve as useful tools in biological experiments aimed to further elucidate native ADP-ribosylation. In this review, we will discuss the synthetic studies on mono-ADP-ribosylated proteins and oligo-ADP-ribose chains. Future possible synthetic targets and upcoming new methods for the synthesis of these molecules are also included.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Humans; Molecular Conformation; Peptides; Protein Processing, Post-Translational
PubMed: 31112180
DOI: 10.1039/c9ob00501c -
Nature Mar 2020The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells. After binding to...
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. 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 factors. 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 residues. 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.
Topics: ADP-Ribosylation; Allosteric Regulation; Amino Acid Motifs; Amino Acid Sequence; Animals; Biocatalysis; Carrier Proteins; Catalytic Domain; DNA Damage; HEK293 Cells; Humans; Models, Molecular; Mutation; NAD; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Sea Anemones
PubMed: 32028527
DOI: 10.1038/s41586-020-2013-6 -
Open Biology Apr 2019ADP-ribosylation (ADPr) is a reversible post-translational modification of proteins, which controls major cellular and biological processes, including DNA damage repair,... (Review)
Review
ADP-ribosylation (ADPr) is a reversible post-translational modification of proteins, which controls major cellular and biological processes, including DNA damage repair, cell proliferation and differentiation, metabolism, stress and immune responses. In order to maintain the cellular homeostasis, diverse ADP-ribosyl transferases and hydrolases are involved in the fine-tuning of ADPr systems. The control of ADPr network is vital, and dysregulation of enzymes involved in the regulation of ADPr signalling has been linked to a number of inherited and acquired human diseases, such as several neurological disorders and in cancer. Conversely, the therapeutic manipulation of ADPr has been shown to ameliorate several disorders in both human and animal models. These include cardiovascular, inflammatory, autoimmune and neurological disorders. Herein, we summarize the recent findings in the field of ADPr, which support the impact of this modification in human pathophysiology and highlight the curative potential of targeting ADPr for translational and molecular medicine.
Topics: ADP Ribose Transferases; ADP-Ribosylation; DNA Repair; Humans; Models, Biological; Neoplasms; Nervous System Diseases; Protein Processing, Post-Translational; Proteins; Signal Transduction
PubMed: 30991935
DOI: 10.1098/rsob.190041