Purification of Human PARP-1 and PARP-1 Domains from Escherichia coli for Structural and Biochemical Analysis

Langelier, Marie-France; Planck, Jamie L.; Servent, Kristin M.; Pascal, John M.

doi:10.1007/978-1-61779-270-0_13

Cited by 90 publications

(77 citation statements)

References 8 publications

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“…Our objective was to obtain a comparable measure of PARP inhibitor potencies for many PARP family members using an assay of enzymatic inhibition. First, we validated a previously published method 22 to ensure unbiased addition of biotin-labeled NAD + onto PARP substrate proteins and onto growing PAR polymers (Supporting Information, Figure S1). Next, we determined the K M values for NAD + turnover by PARP1–4, the two tankyrases, and five members of the mono-ADP-ribosyltransferase subfamily (PARP6−16; Table 1, Supporting Information, Table S1 and Figure S2).…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

et al. 2016

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Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

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Section: Resultsmentioning

confidence: 99%

Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

et al. 2016

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“…(44). Briefly, the proteins were expressed in One Shot TOP10 E. coli cells according to the manufacture's protocol by inducing with 1 mM IPTG, 0.1% arabinose and 0.5 mM ZnCl 2 for 18 h at 15°C.…”

Section: Methodsmentioning

confidence: 99%

Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair

Prasad

Horton

Chastain

et al. 2014

Nucleic Acids Research

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Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme in mammalian cells. The enzyme synthesizes polymers of ADP-ribose from the coenzyme NAD+ and plays multifaceted roles in cellular responses to genotoxic stress, including DNA repair. It had been shown that mouse fibroblasts treated with a DNA methylating agent in combination with a PARP inhibitor exhibit higher cytotoxicity than cells treated with methylating agent alone. This lethality of the PARP inhibitor is dependent on apurinic/apyrimidinic (AP) sites in the DNA and the presence of PARP-1. Here, we show that purified PARP-1 is capable of forming a DNA-protein cross-link (DPC) by covalently attaching to the AP site. This DPC formation is specific to the presence of the natural AP site in DNA and is accompanied by a single-strand DNA incision. Cellular studies confirm the formation of PARP-1 DPCs during alkylating agent-induced base excision repair (BER) and formation of DPCs is enhanced by a PARP inhibitor. Using an N-terminal and C-terminal truncated PARP-1 we show that a polypeptide fragment comprising the zinc 3 and BRCT sub-domains is sufficient for DPC formation. The covalent attachment of PARP-1 to AP site-containing DNA appears to be a suicidal event when BER is overwhelmed or disrupted.

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“…In contrast to the abundance of structural and functional data describing PAR synthesis [1][2][3][4] , our present understanding of the PAR-degradation pathway is comparatively poor 5,6 . Canonical PARG is a highly conserved protein found in organisms ranging from protozoa to humans 7 .…”

mentioning

confidence: 90%

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

Dunstan¹,

Barkauskaite

Lafite

et al. 2012

Nat Commun

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Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DnA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1′′-2′) ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodaninecontaining mammalian PARG inhibitor RBPI-3. our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors.

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Purification of Human PARP-1 and PARP-1 Domains from Escherichia coli for Structural and Biochemical Analysis

Cited by 90 publications

References 8 publications

Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

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