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

ABSTRACTPoly(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 a combination of single-molecule techniques including magnetic tweezers and atomic force microscopy, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-picoNewton mechanical forces. Decondensation by high force proceeds through a series of discrete increases in extension, indicating that PARP1 stabilizes loops of DNA. This model is supported by DNA braiding experiments which show that PARP1 can bind at the intersection of two separate DNA molecules. 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 establish a mechanism for PARP1 in the organization of chromatin structure.

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Single Molecule Fluorescence and Atomic Force Microscopy Studies of DNA Repair

Biophysical Journal ◽

10.1016/j.bpj.2016.11.061 ◽

2017 ◽

Vol 112 (3) ◽

pp. 7a

Author(s):

Dorothy Erie ◽

Kira Bradford ◽

Hunter Wilkins ◽

Jacqueline Bower ◽

Zimeng Wang ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Dna Repair ◽

Single Molecule ◽

Single Molecule Fluorescence ◽

Force Microscopy ◽

Atomic Force

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Polyethylene glycol and divalent salt-induced DNA reentrant condensation revealed by single molecule measurements

Soft Matter ◽

10.1039/c5sm00619h ◽

2015 ◽

Vol 11 (19) ◽

pp. 3927-3935 ◽

Cited By ~ 15

Author(s):

Chao Cheng ◽

Jun-Li Jia ◽

Shi-Yong Ran

Keyword(s):

Atomic Force Microscopy ◽

Polyethylene Glycol ◽

Single Molecule ◽

Magnetic Tweezers ◽

Dna Condensation ◽

Peg 6000 ◽

Force Microscopy ◽

Molecular Weights ◽

Atomic Force ◽

Peg 600

In this study, we investigated the DNA condensation induced by polyethylene glycol (PEG) with different molecular weights (PEG 600 and PEG 6000) in the presence of NaCl or MgCl2 by using magnetic tweezers (MT) and atomic force microscopy (AFM).

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Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

Nanomaterials ◽

10.3390/nano11092424 ◽

2021 ◽

Vol 11 (9) ◽

pp. 2424

Author(s):

Lyan M. van der Sleen ◽

Katarzyna M. Tych

Keyword(s):

Mechanical Properties ◽

Atomic Force Microscopy ◽

Optical Tweezers ◽

Single Molecule ◽

Short Range ◽

Force Spectroscopy ◽

Magnetic Tweezers ◽

Single Molecule Force Spectroscopy ◽

Force Microscopy ◽

Atomic Force

The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.

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

Science Advances ◽

10.1126/sciadv.abf3641 ◽

2021 ◽

Vol 7 (33) ◽

pp. eabf3641

Author(s):

Nicholas A. W. Bell ◽

Philip J. Haynes ◽

Katharina Brunner ◽

Taiana Maia de Oliveira ◽

Maria M. Flocco ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Transcription Regulation ◽

Single Molecule ◽

Chromatin Structure ◽

Parp Inhibitors ◽

Chromatin Organization ◽

Mechanical Forces ◽

Dna Loops ◽

Condensation Activity

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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Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

Nature Methods ◽

10.1038/nmeth.1218 ◽

2008 ◽

Vol 5 (6) ◽

pp. 491-505 ◽

Cited By ~ 1393

Author(s):

Keir C Neuman ◽

Attila Nagy

Keyword(s):

Atomic Force Microscopy ◽

Optical Tweezers ◽

Single Molecule ◽

Force Spectroscopy ◽

Magnetic Tweezers ◽

Single Molecule Force Spectroscopy ◽

Force Microscopy ◽

Atomic Force ◽

Spectroscopy Optical

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Atomic Force Microscopy (AFM) for Topography and Recognition Imaging at Single Molecule Level

Encyclopedia of Biophysics ◽

10.1007/978-3-642-16712-6_496 ◽

2013 ◽

pp. 102-112

Author(s):

Memed Duman ◽

Andreas Ebner ◽

Christian Rankl ◽

Jilin Tang ◽

Lilia A. Chtcheglova ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Single Molecule ◽

Single Molecule Level ◽

Force Microscopy ◽

Atomic Force ◽

Recognition Imaging

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Atomic Force Microscopy Investigation of the Interactions between the MCM Helicase and DNA

Materials ◽

10.3390/ma14030687 ◽

2021 ◽

Vol 14 (3) ◽

pp. 687

Author(s):

Amna Abdalla Mohammed Khalid ◽

Pietro Parisse ◽

Barbara Medagli ◽

Silvia Onesti ◽

Loredana Casalis

Keyword(s):

Atomic Force Microscopy ◽

Nucleic Acid ◽

Single Molecule ◽

Protein Complex ◽

The Other ◽

Contour Length ◽

Force Microscopy ◽

Atomic Force ◽

Mcm Complex ◽

Dna Double Helix

The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from Methanothermobacter thermautotrophicus by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. We discriminated between two modes of interaction, one in which the protein induces a sharp bend in the DNA, and one where there is no bending. We found that the presence of the MCM complex also affects the DNA contour length. A possible interpretation of the observed behavior is that in one case the hexameric ring encircles the dsDNA, while in the other the nucleic acid wraps on the outside of the ring, undergoing a change of direction. We confirmed this topographical assignment by testing two mutants, one affecting the N-terminal β-hairpins projecting towards the central channel, and thus preventing DNA loading, the other lacking an external subdomain and thus preventing wrapping. The statistical analysis of the distribution of the protein complexes between the two modes, together with the dissection of the changes of DNA contour length and binding angle upon interaction, for the wild type and the two mutants, is consistent with the hypothesis. We discuss the results in view of the various modes of nucleic acid interactions that have been proposed for both archaeal and eukaryotic MCM complexes.

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