Quantitation of DNA-Binding Affinity Using Tethered Particle Motion

Henneman, Bram; Heinsman, Joost; Battjes, J.A.; Dame, Remus T.

doi:10.1007/978-1-4939-8675-0_14

Cited by 13 publications

(17 citation statements)

References 39 publications

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“…3H), indicating reduced Brownian motion of the bead-bound GT-DNA end. The decreased RMSD is consistent with MutSα-induced DNA bending (41,83,86). A peak in the RMSD distribution at the position of free DNA remains detectable, which indicates that not all of the DNA is bound by MutSα.…”

Section: Mutsα and Mutlα Form Multimeric Complexes On Mismatched Dnasupporting

confidence: 56%

“…Tethered Particle Motion Assay. Tethered particle motion (TPM) experiments measure changes in DNA configurations that result in changes in the DNA end-to-end distances, such as protein-induced DNA bending, DNA looping, or wrapping, by monitoring the Brownian motion of a bead on the end of a surface-tethered DNA fragment (83)(84)(85). Experimental details of the TPM measurements are in the SI Appendix.…”

Section: Methodsmentioning

confidence: 99%

“…Given the finding of DNA shortening from the AFM experiments, we sought independent in-solution evidence for MutSα-MutLα-induced DNA compaction using tethered particle motion (TPM) experiments (83)(84)(85). For the TPM experiments, we use a 550-bp DNA substrate with a single central GT mismatch (or a GC for homoduplex DNA) tethered to a surface by one end and with a bead attached to the other end (Fig.…”

Section: Mutsα and Mutlα Form Multimeric Complexes On Mismatched Dnamentioning

confidence: 99%

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Dynamic human MutSα–MutLα complexes compact mismatched DNA

Bradford

Wilkins

Hao

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure–function properties of these obligate MutSα–MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα–MutLα–DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS–MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

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Section: Mutsα and Mutlα Form Multimeric Complexes On Mismatched Dnasupporting

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Section: Mutsα and Mutlα Form Multimeric Complexes On Mismatched Dnamentioning

confidence: 99%

See 1 more Smart Citation

Dynamic human MutSα–MutLα complexes compact mismatched DNA

Bradford

Wilkins

Hao

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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show abstract

“…By manipulation of microbeads, single-molecule force spectroscopy techniques revealed the mechanical properties of biomolecules such as DNA or RNA with unprecedented detail ( 2 , 3 , 4 , 5 ). In addition, interactions with proteins such as DNA compaction by histones in eukaryotic chromatin ( 6 , 7 , 8 , 9 , 10 , 11 ) and prokaryotic architectural proteins ( 12 , 13 , 14 , 15 , 16 ), supercoiling ( 17 , 18 , 19 , 20 ), and repair processes ( 21 , 22 , 23 ) were extensively studied with magnetic tweezers (MT), optical tweezers (OT), acoustic force spectroscopy (AFS) ( 24 , 25 , 26 ), or tethered particle motion (TPM) ( 13 , 27 , 28 ). These bead manipulation techniques have also been used to quantify the mechanical properties of other biological structures such as extracellular protein collagen ( 29 , 30 , 31 ) or even entire cells ( 32 ).…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Brouwer

Hermans

Noort

2020

Biophysical Journal

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Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead's position with nanometer accuracy in three dimensions but is computationally expensive. Realtime multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 mm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.

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“…By manipulation of microbeads, single-molecule force spectroscopy techniques revealed the mechanical properties of biomolecules such as DNA or RNA with unprecedented detail [2][3][4][5]. In addition, the interactions with proteins, like the DNA compaction by histones in eukaryotic chromatin [6][7][8][9][10][11] and prokaryotic architectural proteins [12][13][14][15][16], supercoiling [17][18][19][20], and repair processes [21][22][23] were extensively studied with magnetic tweezers (MT) or optical tweezers (OT), Acoustic Force Spectroscopy (AFS) [24][25][26] or tethered particle motion (TPM) [13,27,28]. These bead manipulation techniques have also been used to quantify the mechanical properties of other biological structures, such as extracellular protein collagen [29][30][31], or even entire cells [32].…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

Brouwer

Hermans

Noort

2019

Preprint

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Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in arange of over 10 µm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required. SignificanceMicrobeads are often used in biophysical single-molecule manipulation experiments and accurately tracking their position in 3 dimensions is key for quantitative analysis. Holographic imaging of these beads allows for multiplexing bead tracking but image analysis can be a limiting factor. Here we present a 3D tracking algorithm based on Fast Fourier Transforms that is fast, has nanometric precision, is robust against common artifacts and is accurate over 10's of micrometers. We show its real-time application for magnetic tweezers based force spectroscopy on more than 100 chromatin fibers in parallel, but we anticipate that many other bead-based biophysical essays can benefit from this simple and robust 3 phasor algorithm.

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Quantitation of DNA-Binding Affinity Using Tethered Particle Motion

Cited by 13 publications

References 39 publications

Dynamic human MutSα–MutLα complexes compact mismatched DNA

Dynamic human MutSα–MutLα complexes compact mismatched DNA

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

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