Single-Molecule Analysis of Replication Protein A-DNA Interactions

Fletcher E Bain; Laura A Fischer; Ran Chen; Marc S Wold

doi:10.1016/bs.mie.2017.11.016

Back

Single-Molecule Analysis of Replication Protein A-DNA Interactions

Journal article

Peer reviewed

Single-Molecule Analysis of Replication Protein A-DNA Interactions

Fletcher E Bain, Laura A Fischer, Ran Chen and Marc S Wold

Methods in enzymology, Vol.600, pp.439-461

2018

DOI: 10.1016/bs.mie.2017.11.016

PMID: 29458769

View Online

Abstract

Replication protein A (RPA) is a highly conserved, eukaryotic ssDNA-binding protein essential for genome stability. RPA interacts with ssDNA and with protein partners to coordinate DNA replication, repair, and recombination. Single-molecule analysis of RPA-DNA interactions is leading to a better understanding of the molecular interactions and dynamics responsible for RPA function in cells. Here, we first describe how to express, purify, and label RPA. We then describe how to prepare materials and carry out single-molecule experiments examining RPA-DNA interactions using total internal reflection fluorescence microscopy (TIRFM). Finally, the last section describes how to analyze TIRFM data. This chapter will focus on human RPA. However, these methods can be applied to RPA homologs from other species.

Recombinant Proteins - metabolism

Staining and Labeling - methods

Fluorescent Dyes - chemistry

Recombinational DNA Repair

DNA, Single-Stranded - metabolism

Recombinant Proteins - chemistry

Replication Protein A - isolation & purification

Replication Protein A - metabolism

Image Processing, Computer-Assisted - methods

Single Molecule Imaging - methods

DNA-Binding Proteins - isolation & purification

DNA-Binding Proteins - chemistry

DNA, Single-Stranded - chemistry

Microscopy, Fluorescence - instrumentation

DNA-Binding Proteins - metabolism

Recombinant Proteins - isolation & purification

Video Recording - instrumentation

Single Molecule Imaging - instrumentation

Microscopy, Fluorescence - methods

Replication Protein A - chemistry

Video Recording - methods

Protein Binding

Details

Title: Subtitle: Single-Molecule Analysis of Replication Protein A-DNA Interactions
Creators: Fletcher E Bain - University of Iowa Carver College of Medicine, Iowa City, IA, United States
Laura A Fischer - University of Iowa Carver College of Medicine, Iowa City, IA, United States
Ran Chen - Washington University, St. Louis, MO, United States
Marc S Wold - University of Iowa Carver College of Medicine, Iowa City, IA, United States. Electronic address: marc-wold@uiowa.edu
Resource Type: Journal article
Publication Details: Methods in enzymology, Vol.600, pp.439-461
Publisher: United States
DOI: 10.1016/bs.mie.2017.11.016
PMID: 29458769
ISSN: 0076-6879
eISSN: 1557-7988
Grant note: T32 GM008365 / NIGMS NIH HHS P30 CA086862 / NCI NIH HHS
Language: English
Date published: 2018
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024418202771

Metrics

62 Record Views

12 Times Cited - Web of Science

See more details