Quantification of Poly(ADP-Ribose) In Vitro: Determination of the ADP-Ribose Chain Length and Branching Pattern

Alvarez-Gonzalez, Rafael; Jacobson, Myron K.

doi:10.1007/978-1-61779-270-0_2

Cited by 5 publications

(4 citation statements)

References 15 publications

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“…The measured molecular mass corresponded to polymers lacking a terminal ribose phosphate or a ribose bisphosphate group. This is consistent with literature findings that alkaline release of pADPr from proteins can result in three polymer isoforms: complete polymer, polymer lacking the terminal ribose phosphate, and polymer missing the terminal ribose bisphosphate group [51]. …”

Section: Resultssupporting

confidence: 93%

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Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

Tan¹,

Krukenberg²,

Mitchison³

2012

Analytical Biochemistry

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Poly(ADP-ribose) [pADPr] is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD+ by poly(ADP-ribose) polymerases [PARPs] and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. PADPr is involved in many cellular processes and exerts biological function through covalent modification and non-covalent binding to specific proteins. Very little is known about molecular recognition and structure activity relationships for non-covalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.

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Section: Resultssupporting

confidence: 93%

“…The prevailing method to identify pADPr size is to run a high resolution PAGE and visualize the resolved polymers by radioactivity, immunoblotting, or silver staining [51; 60; 61; 62; 63]. Bromophenol blue and xylene cyanol were used as size markers because they co-migrate with ~8 and 20 mers, respectively [59].…”

Section: Discussionmentioning

confidence: 99%

Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

Tan¹,

Krukenberg²,

Mitchison³

2012

Analytical Biochemistry

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“…We obtained fractions of labelled PAR molecules consisting of approximately 10, 20, 30, 40 and 50 ADP-ribose units displaying a distinct size distribution. It is important to note that the indicated sizes are approximate numbers based on the size markers Orange G (corresponding to an ADP-ribose monomer), bromophenol blue (corresponding to an ADP-ribose 8-mer), and xylene cyanol (corresponding to an ADP-ribose 20-mer) as published previously ( 65 ). Assuming a maximum chain length of ∼50 ADP-ribose units and a branching frequency of ∼1%, it was estimated that PAR WT predominantly consisted of linear PAR molecules, whereas the corresponding PAR HB fraction exhibited ∼7–8 branching points per molecule.…”

Section: Resultsmentioning

confidence: 94%

Influence of chain length and branching on poly(ADP-ribose)–protein interactions

Löffler

Krüger

Zirak

et al. 2023

Nucleic Acids Research

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Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR–protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53—and potentially also APLF—can form complex multivalent PAR–protein structures. In conclusion, our study gives detailed and quantitative insight into PAR–protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.

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“…the acceptor amino acid residue) and different degrees of occupancy (i.e. the molecular stoichiometry of the protein–PARylation reaction or the number of ADP-ribose chains/polypeptide unit) ( 115 ). However, the underlying determinants that modulate PARP-1 activity and PAR complexity in various physiological contexts remain largely unexplored.…”

Section: Structure–function Relationships Of Parpsmentioning

confidence: 99%

The dynamic process of covalent and non-covalent PARylation in the maintenance of genome integrity: a focus on PARP inhibitors

Beneyton,

Nonfoux,

Gagné

et al. 2023

NAR Cancer

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Poly(ADP-ribosylation) (PARylation) by poly(ADP-ribose) polymerases (PARPs) is a highly regulated process that consists of the covalent addition of polymers of ADP-ribose (PAR) through post-translational modifications of substrate proteins or non-covalent interactions with PAR via PAR binding domains and motifs, thereby reprogramming their functions. This modification is particularly known for its central role in the maintenance of genomic stability. However, how genomic integrity is controlled by an intricate interplay of covalent PARylation and non-covalent PAR binding remains largely unknown. Of importance, PARylation has caught recent attention for providing a mechanistic basis of synthetic lethality involving PARP inhibitors (PARPi), most notably in homologous recombination (HR)-deficient breast and ovarian tumors. The molecular mechanisms responsible for the anti-cancer effect of PARPi are thought to implicate both catalytic inhibition and trapping of PARP enzymes on DNA. However, the relative contribution of each on tumor-specific cytotoxicity is still unclear. It is paramount to understand these PAR-dependent mechanisms, given that resistance to PARPi is a challenge in the clinic. Deciphering the complex interplay between covalent PARylation and non-covalent PAR binding and defining how PARP trapping and non-trapping events contribute to PARPi anti-tumour activity is essential for developing improved therapeutic strategies. With this perspective, we review the current understanding of PARylation biology in the context of the DNA damage response (DDR) and the mechanisms underlying PARPi activity and resistance.

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Quantification of Poly(ADP-Ribose) In Vitro: Determination of the ADP-Ribose Chain Length and Branching Pattern

Cited by 5 publications

References 15 publications

Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

Influence of chain length and branching on poly(ADP-ribose)–protein interactions

The dynamic process of covalent and non-covalent PARylation in the maintenance of genome integrity: a focus on PARP inhibitors

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