Book chapter
Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution
Methods in enzymology, pp.201-232
Methods in Enzymology, Elsevier
01/01/2018
DOI: 10.1016/bs.mie.2017.12.008
PMCID: PMC6033278
PMID: 29458759
Abstract
Human RAD51 promotes accurate DNA repair by homologous recombination and is involved in protection and repair of damaged DNA replication forks. The active species of RAD51 and related recombinases in all organisms is a nucleoprotein filament assembled on single-stranded DNA (ssDNA). The formation of a nucleoprotein filament competent for the recombination reaction, or for DNA replication support, is a delicate and strictly regulated process, which occurs through filament nucleation followed by filament extension. The rates of these two phases of filament formation define the capacity of RAD51 to compete with the ssDNA-binding protein RPA, as well as the lengths of the resulting filament segments. Single-molecule approaches can provide a wealth of quantitative information on the kinetics of RAD51 nucleoprotein filament assembly, internal dynamics, and disassembly. In this chapter, we describe how to set up a single-molecule total internal reflection fluorescence microscopy experiment to monitor the initial steps of RAD51 nucleoprotein filament formation in real-time and at single-monomer resolution. This approach is based on the unique, stretched-ssDNA conformation within the recombinase nucleoprotein filament and follows the efficiency of Forster resonance energy transfer (EFRET) between two DNA-conjugated fluorophores. We will discuss the practical aspects of the experimental setup, extraction of the FRET trajectories, and how to analyze and interpret the data to obtain information on RAD51 nucleation kinetics, the mechanism of nucleation, and the oligomeric species involved in filament formation.
Details
- Title: Subtitle
- Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution
- Creators
- Shyamal Subramanyam - Roy J. and Lucille A. Carver College of MedicineColin D. Kinz-Thompson - Columbia UniversityRuben L. Gonzalez - Columbia UniversityMaria Spies - Roy J. and Lucille A. Carver College of Medicine
- Contributors
- A Malkova (Editor)M Spies (Editor) - University of Iowa, Biochemistry and Molecular Biology
- Resource Type
- Book chapter
- Publication Details
- Methods in enzymology, pp.201-232
- Series
- Methods in Enzymology
- DOI
- 10.1016/bs.mie.2017.12.008
- PMID
- 29458759
- PMCID
- PMC6033278
- NLM abbreviation
- Methods Enzymol
- eISSN
- 1557-7988
- ISSN
- 0076-6879
- Publisher
- Elsevier; SAN DIEGO
- Number of pages
- 32
- Grant note
- R01 GM119386; R01 GM084288; R01 GM108617; T32 GM008281 / NIGMS NIH HHS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS) P30 CA086862 / NCI NIH HHS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI) P30CA086862 / NATIONAL CANCER INSTITUTE; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI) R01GM108617 / NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS)
- Language
- English
- Date published
- 01/01/2018
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984293075002771
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