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. 2017:1608:231-253.
doi: 10.1007/978-1-4939-6993-7_16.

Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin

Maggie H Chassé  1   2 , Uma M Muthurajan  1 , Nicholas J Clark  3 , Michael A Kramer  4 , Srinivas Chakravarthy  5 , Thomas Irving  5 , Karolin Luger  6   7   8
Affiliations

Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin

Maggie H Chassé et al. Methods Mol Biol. 2017.

Abstract

Poly (ADP-Ribose) Polymerase I (PARP-1) is a first responder to DNA damage and participates in the regulation of gene expression. The interaction of PARP-1 with chromatin and DNA is complex and involves at least two different modes of interaction. In its enzymatically inactive state, PARP-1 binds native chromatin with similar affinity as it binds free DNA ends. Automodification of PARP-1 affects interaction with chromatin and DNA to different extents. Here we describe a series of biochemical and biophysical techniques to quantify and dissect the different binding modes of PARP-1 with its various substrates. The techniques listed here allow for high throughput and quantitative measurements of the interaction of different PARP-1 constructs (inactive and automodified) with chromatin and DNA damage models.

Keywords: Analytical ultracentrifugation; Atomic force microscopy; Electrophoretic mobility shift assays; HI-FI FRET; Job plot; Multi-angle light scattering; PARP-1; Small angle X-ray scattering.

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Figures

Figure 1
Figure 1. PARP-1 – chromatin interactions analyzed by native PAGE
A. PARP-1 is a multi-domain protein. The N-Parp construct used here is composed of residues 1–486. B. Schematic of mononucleosomes and trinucleosomes used in assays discussed here. Nuc146 and NLE-Tri do not have exposed DNA linker ends, while Nuc165 and Nuc207 do. C. Schematic for preparing automodified and ‘mock-automodified’ PARP-1. Pre-quenching with a Parp inhibitor (PJ34) prior to addition of DNA and NAD+ inhibits the NAD+ binding site in PARP-1. Automodified PARP-1 samples are quenched with PJ34 after incubation with DNA and NAD+. Each probe has the same reaction components to ensure binding affinity differences are due to PARylation. D. PAGE analysis of Alexa-488 fluorescently labeled PARP-1. PARP-1, pre-quenched PARP-1 (pQ), and automodified PARP-1 (AM) were run on a denaturing PAGE. Left: Protein stain of 1 µg unlabeled PARP-1 reactions. Lane 1, molecular weight marker; Lane 2, PARP-1 alone; Lane 3, pre-quenched PARP-1; Lane 4, automodified PARP-1. Middle: Protein stain of 1 µg Alexa488 labeled PARP-1. Lane 1, molecular weight marker; Lane 2, PARP-1 alone; Lane 3, pre-quenched PARP-1; Lane 4, automodified PARP-1. Right: Cy2 (ex/em: 473/520 nm) typhoon scan of 0.25 µg Alexa488 labeled PARP-1. Lane 1, pre-quenched PARP-1; Lane 2, automodified PARP-2; Lane 3, molecular weight marker. Both unlabeled and labeled PARP-1 samples show a single homogenous band (with less than 5% degradation), while automodification (AM) creates a heterogeneous smear or shifted band on the gel due to PARylation. E. EMSA analysis of PARP-1, unlabeled and Alexa488 labeled, with unlabeled and Atto-547N labeled mononucleosomes. Nuc165 was incubated with PARP-1 (0.5, 1, and 1.5-fold excess) and analyzed on a 5% TBE-PAGE. Gels were stained as indicated. FL-PARP-1 binding to mononucleosomes results in a supershifted band. Addition of a fluorescent label to PARP-1 and Nuc165 does not effect this interaction. Lane 1, molecular weight marker; Lane 2, 165 bp DNA; Lane 3, unlabeled Nuc165; Lane 4–6, increasing titrations of unlabeled PARP-1 with unlabeled Nuc165; Lane 7, Atto-647N labeled Nuc165; Lane 8–10, increasing titration of Alexa488 labeled PARP-1 with Atto-647N Nuc165. F. EMSA analysis of N-Parp with NLE-Tri. NLE-Tri was incubated with N-Parp (0.8, 1, 1.2, 1.4, 1.6, and 1.8-fold excess) and analyzed on 1% agarose. Gels were stained as indicated. N-Parp binding to trinucleosomes resulted in band of slower mobility. Lane 1, Molecular weight marker (1kb); Lane 2, NLE-Tri alone; Lanes 3–8, increasing titration of N-Parp (0.8–1.8) with NLE-Tri; Lane 9, NLE-Trimer DNA (561 bp). Samples from lanes 2 and 6 were used in the AUC experiments in Figure 3D.
Figure 2
Figure 2. HIFI-FRET binding & Job plot stoichiometry assays
A. Schematic of HIFI-FRET binding assays (top) and representative FRET plate typhoon scans (bottom). Donor probe (e.g. PARP-1) is kept constant while acceptor ligand (e.g. Nuc165) is titrated. Acceptor only (acceptor + buffer) in the top two rows; donor only (donor + only) in the first two wells in the FRET series. Interaction between the two binding partners yields FRET. B. A representative binding curve of PARP-1 binding mononucleosomes with and without DNA linker ends. PARP-1 binds Nuc146 (□, dashed line) with weak affinity (> 500 nM) and no saturatable plateau. PARP-1 binds Nuc165 (●) with low nanomolar affinity (~2 nM). C. Representative binding curve for Pre-Quenched PARP-1 (pQ) (●) and AM-PARP-1 (□, dashed line) to NLE-Tri. PARP-1 binds tightly to NLE-Tri (~5 nM); the affinity weakens 20-fold when PARP-1 is automodified (~101 nM). Note that panel 2C is reused from a previous publication (3). D. Schematic of Job plot stoichiometry experimental set up (top) and representative plate typhoon scans (bottom). The donor probe (here, PARP-1) is incrementally increased while acceptor ligand (here, Nuc165) is incrementally decreased keeping the overall molar ratio equal to one. Interaction between the two binding partners yields FRET signal. E. Representative Job plot stoichiometry curve for PARP-1 binding double stranded DNA (●) and mononucleosomes (□). The dashed lines indicate the peak of each curve and the ideal molar ratio. At a peak of 0.5, PARP-1 binds Nuc165 in a 1 to 1 stoichiometry. DNA binds two PARP-1 molecules (peak of 0.66).
Figure 3
Figure 3. Solution state analysis of PARP-1 binding mono- and tri-nucleosomes
A. Analysis of PARP-1 binding mononucleosomes via size exclusion coupled multi-angle light scattering. Nuc207 forms a 1:1 complex with PARP-1 even when excess PARP-1 is added. The molecular weights for the various complexes derived are listed in the table. Note that panel 3A figure and table are reused from (5). B. Analysis of PARP-1 by AUC. Sedimentation velocity profile for N-Parp (□) indicates an Savg value of ~3.2S and ~5S for full length PARP-1 (○). The vertical nature of the G(s) plot indicates homogeneity of the protein samples. C. Analysis of PARP-1 interaction with mononucleosomes via AUC. Sedimentation velocity profile for Nuc165 (○, gray) gives rise to an Savg of ~12S, complex formation with N-Parp (□) results in a shift of the complex Savg to ~13S; and full length PARP-1 (Δ) interaction with nucleosomes shifts the Savg to ~14S. D. Analysis of N-Parp tri-nucleosome complexes via AUC. Sedimentation velocity profile for NLE-Tri (●, gray) has an Savg of ~17S, interaction with N-Parp (■) results in an Savg of ~21S.
Figure 4
Figure 4. Atomic force microscopy of trinucleosomes and PARP-1 trinucleosome complexes
A, B, C. AFM of NLE-Tri and PARP-1 NLE-Tri complexes. 1–1.5 µm scans are shown here. A. NLE-Tri alone; B. NLE-Tri + PARP-1; C. NLE-Tri + automodified PARP-1 (AM-PARP-1). Height profiles of selected particles are shown underneath each image. In the absence of PARP-1, trinucleosomes have a height profile of 1.5–2 nm but in the presence of PARP-1, the trinucleosomes are compacted (height profiles increase to 3–5 nm). Compaction is alleviated by automodification of PARP-1 and release from chromatin (height profiles similar to no PARP-1). Note that panels 4A-C are reused from a previous publication (3).
Figure 5
Figure 5. Small angle x-ray scattering (SAXS) of N-Parp – mononucleosome complex
A. SAXS data for the mononucleosome. Left panel - Guinier plot for data acquired for Nuc165 indicates a radius of gyration (Rg) of 47 Å. Right panel - P (R) function plot for Nuc165 indicates a maximum dimension (Dmax) of ~157 Å. B. SAXS data for N-Parp mononucleosome complex. Left panel - Guinier plot for Nuc165-N-Parp complex indicates an Rg of ~55 Å. Right panel - P (R) function plot for mononucleosomes indicates a Dmax of ~194 Å. C. Overlay of the normalized P(R) function plot for Nuc165 (■, gray) and N-Parp+Nuc165 complex (●, black). Shift is indicative of an increase in size potentially indicative of PARP binding.
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