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. 2021 Aug 11;7(33):eabf3641.
doi: 10.1126/sciadv.abf3641. Print 2021 Aug.

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization

Nicholas A W Bell  1   2 , Philip J Haynes  2   3   4 , Katharina Brunner  5   6 , Taiana Maia de Oliveira  7 , Maria M Flocco  7 , Bart W Hoogenboom  2   4 , Justin E Molloy  5
Affiliations

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization

Nicholas A W Bell et al. Sci Adv. .

Abstract

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using single-molecule techniques, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-piconewton mechanical forces. Stepwise decondensation at high force and DNA braiding experiments show that the condensation activity is due to the stabilization of DNA loops by PARP1. PARP inhibitors do not affect the level of condensation of undamaged DNA but act to block condensation reversal for damaged DNA in the presence of NAD+ Our findings suggest a mechanism for PARP1 in the organization of chromatin structure.

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Figures

Fig. 1
Fig. 1. TIRF imaging shows condensation of DNA by PARP1.
(A) Schematic of TIRF microscopy imaging of a single λ-DNA molecule stained with SYTOX Orange. A constant flow was maintained, which stretches out the DNA over a distance close to its contour length. (B) Kymograph showing DNA extension over time. 400 nM PARP1 is added at the time point indicated by the asterisk. (C) Snapshots showing individual image frames at the indicated time points.
Fig. 2
Fig. 2. AFM characterization of PARP1-DNA binding.
(A) AFM images of PARP1 binding to a 4.4-kbp, covalently closed plasmid. Inset: Zoom-in image showing PARP1 bound to plasmid DNA. The presence of bound PARP1 is indicated in green, representing the pixels where the local height exceeded a threshold of ~2 nm above the background. Scale bars, 100 nm. Color scale, 2.5 nm. (B) Images showing PARP1 binding to a 496-bp polymerase chain reaction (PCR) fragment with a SSB approximately one-third of the way along the contour length. The green indicates bound PARP1 that was selected for volume estimation. Scale bars, 50 nm. Color scale, 2.5 nm. (C) Two selected height profiles with corresponding proteins shown in (B). We observe similar height profiles for PARP1 molecules bound to DNA in the presence and absence of 500 nM olaparib. The transparent areas indicate the masked portion of the profiles that contributed to the volume estimate. (D) Histogram of observed PARP1 volumes when bound to the 496-bp PCR fragment.
Fig. 3
Fig. 3. Magnetic tweezers showing PARP1-induced condensation on undamaged DNA.
(A) Schematic of magnetic tweezers showing DNA stretched between the bead and coverslip. The height of the magnets controls the force acting on the bead, and the bead can be rotated by rotating the magnets. (B) DNA construct used for magnetic tweezers with two labeled handles acting as attachment points. (C) Single DNA tethers without nicks are selected by examining the characteristic response of DNA extension to magnet turns. (D) Effect of force ramp on DNA extension in the presence of 400 and 0 nM PARP1. At low forces (<1 pN), PARP1 induces significant compaction of the DNA. The dashed gray line shows a fit of the worm-like chain model to the DNA extension for 0 nM PARP1 with a fixed persistence length of 50 nm. The DNA contour length was a free parameter for the fit and yielded 2.7 μm in good agreement with the expected length of 2.6 μm for the 7.9-kbp DNA.
Fig. 4
Fig. 4. Concentration dependence of DNA condensation by PARP1.
(A) Magnetic tweezers data showing the change in DNA extension at different applied forces. The dashed black line indicates the worm-like chain model prediction for naked DNA. (B) Agarose gel electrophoresis showing titration of PARP1 with 3 nM relaxed, covalently closed 2.7-kbp DNA plasmid. Several bands are visible for the plasmid sample, corresponding to different topoisomers. (C) Force-extension curves measured in the presence and absence of three PARP inhibitors. 400 nM PARP1 concentration was used in each case.
Fig. 5
Fig. 5. Stepwise reversal of PARP1-induced condensation, as observed upon application of high forces.
(A) Traces for 0 and 400 nM PARP1, showing the changes in DNA extension after a step change in force from 0.6 to 2.2 pN. The orange line indicates the result of the step-fitting algorithm used with the arrows showing the positions of individual detected steps. A delay of 0.2 s between the force change and the beginning of step detection was imposed. (B) Histogram of the measured step sizes. Eighty-one steps were detected from a total of 44 traces where the force was stepped from 0.6 to 2.2 pN.
Fig. 6
Fig. 6. Bridging of two DNA molecules by PARP1.
(A) Schematic of experiment—rotating a dual-tethered magnetic bead results in the formation of a DNA braid and a change in measured extension. (B) In the absence of PARP1, rotation of the magnetic bead clockwise (+1 turns) or counterclockwise (−1 turns) results in a reversible change in DNA extension. The three colors represent the data recorded simultaneously from three beads in the field of view. (C) In the presence of 200 nM PARP1, the effect of a one-turn rotation is found to be irreversible, until (D) the flow through of 0 nM PARP1 buffer (at t = 230 s) results in a rapid reversal to original DNA extension. Arrows indicate steps where DNA extension returns to original value.
Fig. 7
Fig. 7. Reversal of PARP1-induced DNA condensation in the presence of NAD+ and DNA damage.
(A) Positions of 14 nick sites on 7.9-kbp DNA section of magnetic tweezers for the nicking endonucleases Nt.BsmAI and Nb.BsmI. (B) Example trace showing the formation of nicks visualized in real time with magnetic tweezers. The DNA is coiled by magnet rotation before addition of nicking endonuclease, which results in rapid uncoiling. (C) Schematic of experiments for measuring the time dependence of condensation. PARP1 is added to damaged DNA before flowing through solution containing NAD+. (D) Force-extension curves showing the change in condensation after adding 400 nM PARP1 followed by adding 1 mM NAD+. (E) Extension, relative to naked DNA, measured at 0.1-pN force as a function of time after flowing through NAD+-containing solution. The graph shows results from two experiments: (i) addition of 1 mM NAD+ (n = 9 magnetic beads) and (ii) addition of 1 mM NAD+ + 10 μM olaparib (n = 4). The error bars show SD.
Fig. 8
Fig. 8. Loop stabilization model for PARP1 condensation of DNA.
(Left) In the absence of protein and at low forces, thermal fluctuations induce the formation of DNA loops. (Right) The multiple DNA binding sites of PARP1 enable bridging to stabilize the loop and thereby reduce the DNA extension.
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