scholarly journals Activation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ezeogo Obaji ◽  
Mirko M. Maksimainen ◽  
Albert Galera-Prat ◽  
Lari Lehtiö
Keyword(s):  
Crystal Structure ◽  
Dna Damage ◽  
Dna Repair ◽  
Conformational Changes ◽  
Regulatory Domain ◽  
Damage Site ◽  
Adp Ribosylation ◽  
Adp Ribosyltransferase ◽  
Dna Ends ◽  
Binding Substrate

AbstractHuman PARP2/ARTD2 is an ADP-ribosyltransferase which, when activated by 5′-phosphorylated DNA ends, catalyses poly-ADP-ribosylation of itself, other proteins and DNA. In this study, a crystal structure of PARP2 in complex with an activating 5′-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, including reorganization of helical fragments, in the PARP2 regulatory domain. A comparison of PARP1 and PARP2 crystal structures reveals how binding to a DNA damage site leads to formation of a catalytically competent conformation. In this conformation, PARP2 is capable of binding substrate NAD+ and histone PARylation factor 1 that changes PARP2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.

scholarly journals Activation of ARTD2/PARP2 by DNA damage induces conformational changes relieving enzyme autoinhibition

2020 ◽  
Author(s):  
Ezeogo Obaji ◽  
Mirko M. Maksimainen ◽  
Albert Galera-Prat ◽  
Lari Lehtiö
Keyword(s):  
Crystal Structure ◽  
Dna Damage ◽  
Dna Repair ◽  
Conformational Changes ◽  
Regulatory Domain ◽  
Damage Site ◽  
Adp Ribosylation ◽  
Adp Ribosyltransferase ◽  
Dna Ends ◽  
Binding Substrate

AbstractHuman ARTD2/PARP2 is an ADP-ribosyltransferase which, when activated by 5’- phosphorylated DNA ends, catalyzes poly-ADP-ribosylation of itself, other proteins and DNA. A crystal structure of ARTD2 in complex with an activating 5’-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, reorganization of helical fragments, in the ARTD2 regulatory domain. Comparison of ARTD1-3 crystal structures reveal how binding to a DNA damage site leads to formation of a catalytically competent conformation capable of binding substrate NAD+ and histone PARylation factor 1 changing the ARTD2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.

scholarly journals HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fa-Hui Sun ◽  
Peng Zhao ◽  
Nan Zhang ◽  
Lu-Lu Kong ◽  
Catherine C. L. Wong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Active Site ◽  
Negative Charge ◽  
Structural Data ◽  
Dna Breaks ◽  
Adp Ribosylation ◽  
Local Conformation ◽  
Key Residues ◽  
Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

The direct interaction of NME3 with Tip60 in DNA repair

2016 ◽  
Vol 473 (9) ◽  
pp. 1237-1245 ◽  
Author(s):  
Ning Tsao ◽  
Ya-Chi Yang ◽  
Yu-Jyun Deng ◽  
Zee-Fen Chang
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Direct Interaction ◽  
Ndp Kinase ◽  
Damage Site ◽  
Site Specific ◽  
Catalytic Function ◽  
Repair Efficiency ◽  
Subsequent Step ◽  
Dna Replication And Repair

Cellular supply of dNTPs via RNR (ribonucleotide reductase) is crucial for DNA replication and repair. It has been shown that DNA-damage-site-specific recruitment of RNR is critical for DNA repair efficiency in quiescent cells. The catalytic function of RNR produces dNDPs. The subsequent step of dNTP formation requires the function of NDP kinase. There are ten isoforms of NDP kinase in human cells. In the present study, we identified NME3 as one specific NDP kinase that interacts directly with Tip60, a histone acetyltransferase, to form a complex with RNR. Our data reveal that NME3 recruitment to DNA damage sites depends on this interaction. Disruption of interaction of NME3 with Tip60 suppressed DNA repair in serum-deprived cells. Thus Tip60 interacts with RNR and NME3 to provide site-specific synthesis of dNTP for facilitating DNA repair in serum-deprived cells which contain low levels of dNTPs.

scholarly journals Serine ADP-ribosylation reversal by the hydrolase ARH3

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Pietro Fontana ◽  
Juan José Bonfiglio ◽  
Luca Palazzo ◽  
Edward Bartlett ◽  
Ivan Matic ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Posttranslational Modification ◽  
Quantitative Proteomics ◽  
Chromatin Regulation ◽  
Adp Ribosylation ◽  
Cellular Processes ◽  
New Form ◽  
Hydrolysis Of ◽  
In Cells

ADP-ribosylation (ADPr) is a posttranslational modification (PTM) of proteins that controls many cellular processes, including DNA repair, transcription, chromatin regulation and mitosis. A number of proteins catalyse the transfer and hydrolysis of ADPr, and also specify how and when the modification is conjugated to the targets. We recently discovered a new form of ADPr that is attached to serine residues in target proteins (Ser-ADPr) and showed that this PTM is specifically made by PARP1/HPF1 and PARP2/HPF1 complexes. In this work, we found by quantitative proteomics that histone Ser-ADPr is reversible in cells during response to DNA damage. By screening for the hydrolase that is responsible for the reversal of Ser-ADPr, we identified ARH3/ADPRHL2 as capable of efficiently and specifically removing Ser-ADPr of histones and other proteins. We further showed that Ser-ADPr is a major PTM in cells after DNA damage and that this signalling is dependent on ARH3.

scholarly journals Serine-linked PARP1 auto-modification controls PARP inhibitor response

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evgeniia Prokhorova ◽  
Florian Zobel ◽  
Rebecca Smith ◽  
Siham Zentout ◽  
Ian Gibbs-Seymour ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Parp Inhibitor ◽  
Cellular Responses ◽  
Vital Role ◽  
Inhibitor Tolerance ◽  
Adp Ribosylation ◽  
Key Sites ◽  
Dependent Modification

AbstractPoly(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.

scholarly journals HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage

2021 ◽  
Author(s):  
Rebecca Smith ◽  
Siham Zentout ◽  
Catherine Chapuis ◽  
Gyula Timinszky ◽  
Sebastien Huet
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Dna Lesions ◽  
End Joining ◽  
Adp Ribosylation ◽  
Damage Response ◽  
Non Homologous End Joining ◽  
Chromatin Relaxation ◽  
The Impact

PARP1 activity is regulated by its cofactor HPF1. The binding of HPF1 on PARP1 controls the grafting of ADP-ribose moieties on serine residues of proteins nearby the DNA lesions, mainly PARP1 and histones. However, the impact of HPF1 on DNA repair regulated by PARP1 remains unclear. Here, we show that HPF1 controls both the number and the length of the ADP-ribose chains generated by PARP1 at DNA lesions. We demonstrate that HPF1-dependent histone ADP-ribosylation, rather than auto-modification of PARP1, triggers the rapid unfolding of the chromatin structure at the DNA damage sites and promotes the recruitment of the repair factors CHD4 and CHD7. Together with the observation that HPF1 contributes to efficient repair both by homologous recombination and non-homologous end joining, our findings highlight the key roles played by this PARP1 cofactor at early stages of the DNA damage response.

scholarly journals Detection of DNA Damage in Prokaryotes by Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End Labeling

2000 ◽  
Vol 66 (3) ◽  
pp. 1001-1006 ◽  
Author(s):  
Forest Rohwer ◽  
Farooq Azam
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Dna Repair ◽  
Uv Light ◽  
Fluorescein Isothiocyanate ◽  
Protein Synthesis Inhibitors ◽  
Dna Damage And Repair ◽  
Dna Damaging Agents ◽  
Bacteriophage Infection ◽  
Dna Ends

ABSTRACT Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3′-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3′-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeonHaloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3′-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.

scholarly journals DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites

2017 ◽  
Vol 217 (2) ◽  
pp. 527-540 ◽  
Author(s):  
Shalaka Chitale ◽  
Holger Richly
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Dna Lesions ◽  
Histone H4 ◽  
Damage Site ◽  
Nucleotide Excision ◽  
Lesion Recognition

Ultraviolet (UV) irradiation triggers the recruitment of DNA repair factors to the lesion sites and the deposition of histone marks as part of the DNA damage response. The major DNA repair pathway removing DNA lesions caused by exposure to UV light is nucleotide excision repair (NER). We have previously demonstrated that the endoribonuclease DICER facilitates chromatin decondensation during lesion recognition in the global-genomic branch of NER. Here, we report that DICER mediates the recruitment of the methyltransferase MMSET to the DNA damage site. We show that MMSET is required for efficient NER and that it catalyzes the dimethylation of histone H4 at lysine 20 (H4K20me2). H4K20me2 at DNA damage sites facilitates the recruitment of the NER factor XPA. Our work thus provides evidence for an H4K20me2-dependent mechanism of XPA recruitment during lesion recognition in the global-genomic branch of NER.

scholarly journals XPA: DNA Repair Protein of Significant Clinical Importance

2020 ◽  
Vol 21 (6) ◽  
pp. 2182 ◽  
Author(s):  
Lucia Borszéková Pulzová ◽  
Thomas A. Ward ◽  
Miroslav Chovanec
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Interactions ◽  
Excision Repair ◽  
Protein A ◽  
Clinical Importance ◽  
Chemotherapeutic Agents ◽  
Dna Lesions ◽  
Response To Treatment ◽  
Damage Site

The nucleotide excision repair (NER) pathway is activated in response to a broad spectrum of DNA lesions, including bulky lesions induced by platinum-based chemotherapeutic agents. Expression levels of NER factors and resistance to chemotherapy has been examined with some suggestion that NER plays a role in tumour resistance; however, there is a great degree of variability in these studies. Nevertheless, recent clinical studies have suggested Xeroderma Pigmentosum group A (XPA) protein, a key regulator of the NER pathway that is essential for the repair of DNA damage induced by platinum-based chemotherapeutics, as a potential prognostic and predictive biomarker for response to treatment. XPA functions in damage verification step in NER, as well as a molecular scaffold to assemble other NER core factors around the DNA damage site, mediated by protein–protein interactions. In this review, we focus on the interacting partners and mechanisms of regulation of the XPA protein. We summarize clinical oncology data related to this DNA repair factor, particularly its relationship with treatment outcome, and examine the potential of XPA as a target for small molecule inhibitors.

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