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Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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生物化学

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Published: August 30, 2024
doi:
10.3791/67251
, ,
1State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy,Nankai University

Summary

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Abstract

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Introduction

Telomeres are protective structures at the ends of chromosomes1,2,3. Telomere erosion during cell division leads to cell senescence and aging, while abnormal elongation of telomeres contributes to cancer4,5. For telomeres to function properly, their replication, elongation, and damage responses must be highly regulated6,7,8. Shelterin, composed of six subunits, plays a central role in telomere protection9,10,11. A deeper understanding of telomeres will provide valuable insights into telomere biology.

TRF1 and TRF2, core subunits of shelterin, are telomeric binding proteins12,13. Both TRF1 and TRF2 bind to the repetitive DNA motif TTAGGG in telomeres via their Myb domains14. They form dimers through their shared TRFH domains, which allow them to encircle telomeric double-stranded DNA and to recruit telomeric proteins15,16,17,18,19. TRF2 is particularly important for the formation of telomeric D-loops and T-loops20,21. Due to their crucial roles in telomere protection, TRF1 and TRF2 have emerged as potential anti-cancer drug targets22,23,24,25.

Significant efforts have been made to investigate the protein-DNA interactions at telomeres. Biochemical methods such as Electrophoretic Mobility Shift Assay (EMSA) and Surface Plasmon Resonance (SPR) have been used to examine binding affinities20,26. Numerous structures of telomeric binding proteins complexed with DNA have been elucidated using cryo-electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR)27,28,29. Super-resolution imaging techniques like stochastic optical reconstruction microscopy (STORM) have revealed TRF2-dependent T-loop formation21. Recently, nanopore sequencing has been developed to profile telomeric sequences4,30,31. These structural insights have greatly enhanced our understanding of telomeric protein-DNA interactions. To further explore the dynamics of telomeric protein-DNA interactions, the development of new technologies is essential.

Single-molecule tools are powerful techniques for exploring protein-DNA interactions at telomere32,33,34. Single-molecule mechanical methods, such as magnetic tweezers, optical tweezers and AFM, have been employed to investigate TRF2-dependent DNA distortion, reveal TRF2-mediated columnar stacking of human telomeric chromatin and observe processive telomerase catalysis, among other applications35,36,37,38,39,40. These methods are particularly useful for probing topological conformations and the kinetics of protein-DNA association and dissociation.

However, the preparation of single-molecule constructs with telomeric repetitive motifs still presents challenges, which limits studies using single-molecule mechanical methods. To address this limitation, we have developed a single-molecule mechanical method to study global protein-DNA interactions on full-length human telomeres41. This method directly extracts telomeric DNA from human cells, circumventing the laborious preparation of artificial telomeric DNA. It facilitates the investigation of kinetic processes on long native telomeres spanning several kilobases.

In this protocol, we provide a detailed description of the steps for probing telomeric protein-DNA interactions using magnetic tweezers, a popular single-molecule mechanical tool42,43,44. We demonstrate how to express and purify telomeric proteins, using TRF2 as an example, and how to prepare telomeric DNA from human cells. Additionally, we show how to set up a single-molecule assay on magnetic tweezers to study telomeric protein-DNA interactions, and we cover the subsequent data analysis of single-molecule experiments. This protocol will benefit researchers in the field of telomere biology and telomere-targeted drug discovery.

Protocol

1. General materials and methods Refer to the Table of Materials file for the salts, chemicals, antibiotics, enzymes, antibodies, and resin materials used in this protocol. Prepare Luria-Bertani (LB) liquid medium, LB agar plates, HEPES buffer, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Phosphate-buffered saline (PBS), Tris-EDTA (TE) buffer according to the recipes from cold spring harbor protocols45,46</s…

Representative Results

Figure 1A illustrates the schematic domains and structures of TRF1 and TRF2, consisting of 439 and 542 amino acids, respectively, which can be expressed in prokaryotic cells. The preparation of TRF1 has been previously described in the literature41. Here, we provide a comprehensive description and representative results of the preparation of TRF2. Figure 1B shows the plasmid map used for expressing TRF2 in E. coli. We evaluated T…

Discussion

This protocol employs magnetic tweezers for the manipulation of TRFs at the single-molecule level57,58,59. We utilize magnetic beads to separate TRFs from genomic DNA fragments. Following restriction digestion, TRFs bind to the magnetic beads, enabling their easy separation from genomic DNA fragments. This approach allows for manipulation using magnetic tweezers, which can effectively trap magnetic beads, unlike optical tweezers…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [Grant 32071227 to Z.Y.], Tianjin Municipal Natural Science Foundation of China (22JCYBJC01070 to Z.Y.), and State Key Laboratory of Precision Measuring Technology and Instruments (Tianjin University) [Grant pilab2210 to Z.Y.].

Materials

Anti-Digoxigenin Roche 11214667001
BfaI New England Biolab (NEB) R0568S
BSA Sigma-Aldrich V900933
CMOS camera  Mikrotron MC1362
CviAII New England Biolab (NEB) R0640S
DIG-11-dUTP Jena Bioscience NU-803-DIGXL
DNA extraction solution G-CLONE EX0108
Dnase I, Rnase-Free, Hc Ea Thermo Fisher Scientific EN0523
dNTP mixture Nanjing Vazyme Biotech Co., Ltd (Vazyme) P032-02
DTT Solarbio D1070
Dynabeads M-270  beads Thermo Fisher Scientific 65305 Streptavidin beads
Dynabeads MyOne beads Thermo Fisher Scientific 65001 Streptavidin beads
Ethanol Tianjin No.6 Chemical Reagent Factory 1083
Glycerol Beijing Hwrkchemical Co,. Ltd SMG66258-1
Imidazole Solarbio II0070
IPTG Solarbio I8070
Isopropanol Tianjin No.6 Chemical Reagent Factory A1079
Kanamycin Thermo Fisher Scientific EN0523
Klenow fragment (3′-5′ exo-) New England Biolab (NEB) M0212S
LabView National Instruments https://www.ni.com/en-us/shop/product/labview.html Graphical programming software 
LiCl Bide Pharmatech Co., Ltd (bidepharm) BD136449
Lysozyme Solarbio L8120-5
MseI New England Biolab (NEB) R0525S
NaCl Shanghai Aladdin C111533
NanoDrop Thermo Fisher Scientific Spectrophotometer
NdeI New England Biolab (NEB) R0111S
Ni NTA Beads 6FF Changzhou Smart-Lifesciences Biotechnology Co.,Ltd SA005025
Nitrocellulose membrane ABclonal RM02801
PMSF Solarbio P8340
Proteinase K Beyotime Biotech Inc (beyotime) ST535-500mg
rCutSmart Buffer New England Biolab (NEB) B6004S
Rnase A Sigma-Aldrich R4875
Sodium acetate SERVA Electrophoresis GmbH 2124902
Sumo protease Beyotime Biotech Inc (beyotime) P2312M
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Gao, H., Liu, Y., Yu, Z. Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers. J. Vis. Exp. (210), e67251, doi:10.3791/67251 (2024).
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