Elsevier

Methods in Enzymology

Volume 592, 2017, Pages 213-257
Methods in Enzymology

Chapter Ten - Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time

https://doi.org/10.1016/bs.mie.2017.03.027Get rights and content

Abstract

Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein–DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair.

Introduction

Experiments studying nucleotide excision repair (NER) proteins using optical imaging in our laboratories usually go through three distinct phases: biochemical analysis (Croteau et al., 2008, Croteau et al., 2006), atomic force microscopy (AFM) (Wang et al., 2006), and fluorescence single-molecule imaging (Hughes et al., 2013, Kad et al., 2010, Kong et al., 2016). First, proteins should be highly purified and exhibit excellent activity. Purification of these proteins often includes a size-exclusion chromatography step to ensure a homogenous preparation of nonaggregated protein, free of contaminating DNA, which is then examined by a variety of bulk biochemistry methods such as fluorescence anisotropy and electrophoretic mobility shift assays for DNA-binding affinities. These proteins are then imaged alone and complexed with DNA substrates using AFM to assess properties such as homogeneity, stability, stoichiometry (Ghodke et al., 2014, Yeh et al., 2012), specificity, and DNA bend angles (Kong et al., 2016). Finally, the dynamic interactions of these proteins with DNA are visualized with the DNA tightrope assay and fluorescence microscopy (Ghodke et al., 2014, Kad et al., 2010, Kong et al., 2016, Kong and Van Houten, 2016). This chapter first gives detailed protocols on preparing defined DNA substrates for analysis by AFM or our tightrope assay. We then discuss how AFM is used to determine specificity, stoichiometry, and DNA bend angles. Finally, we end with a description of our optical DNA tightrope flow cell setup with which we can observe quantum dot (Qdot or QD)-labeled proteins using oblique angle illumination on a total internal reflection fluorescence microscope.

Section snippets

Preparation of Defined Lesion Substrates for AFM and DNA Tightrope Assay

To characterize protein–DNA interactions involving proteins that recognize specific targets, DNA sequences or otherwise, it is important to ensure that an optimal number of target sites exist in the DNA substrate against a vast nonspecific background, such that binding events can be observed efficiently. For DNA repair proteins that carry out damage recognition, a common method to globally induce different types of lesions in a random manner is to subject commercially available λ-DNA to

Atomic Force Microscopy

AFM provides a topographical view of protein–DNA interactions (Fig. 3). Three major sets of data can be obtained from a single protein–DNA experiment: protein specificity for site-specific lesions, as determined by its binding position on a DNA substrate (Fig. 4B and F); the bend angle of DNA at points of specific and nonspecific protein binding or otherwise (Fig. 4C and G); and the stoichiometry of protein binding to DNA substrates as determined by the volume of the complex (Fig. 4A, D, and

Single-Molecule DNA Tightrope Assay

To eliminate the need for constant flow and the potential of surface interactions, we have developed a unique optical platform, based on the ability to anchor both ends of a long DNA molecule on two nearby micron-sized poly-l-lysine-coated silica beads via electrostatic interaction, with the rest of the DNA suspended in between them, forming DNA tightropes (Fig. 5) (Kad et al., 2010). While the procedure involved does not offer the degree of precision and control afforded by the nanofabrication

Conclusions

In summary, we have established a complete laboratory workflow from bulk biochemistry to single-molecule biophysics. The experimental platform detailed in this chapter is well suited for characterization of not just proteins involved in NER, but protein–DNA interactions in general. Specifically, the DNA tightrope assay is straightforward to implement and its versatility allows the technique to be applied to investigate repair pathways such as base excision repair and mismatch repair, as well as

Acknowledgments

This work was made possible through funding from the National Institutes of Health 5R01ES019566 to B.V.H., and 2P30CA047904 to University of Pittsburgh Cancer Institute.

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