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


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.

SUBMITTER: Kong M 

PROVIDER: S-EPMC5656006 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Kong Muwen M   Beckwitt Emily C EC   Springall Luke L   Kad Neil M NM   Van Houten Bennett B  

Methods in enzymology 20170531


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 i  ...[more]

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