Name: Video: Single Molecule Total Internal Reflection Fluorescence Assay for Visualizing CMG-Mediated DNA Unwinding
Uploaded: 2024-09-27T14:20:00
Duration: 3 min 55 s
Description: 167 Views. Single Molecule Total Internal Reflection Fluorescence Assay for Visualizing CMG-Mediated DNA Unwinding

single molecule total internal reflection fluorescence assay for visualizing cmg-mediated dna unwinding

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

DNA replication is tightly regulated, as a cell must duplicate its genome accurately to prevent mutations, disease, or death. Eukaryotic DNA replication is carried out by the replisome complex, which unwinds parental DNA and uses single-stranded DNA (ssDNA) as a template to synthesize new DNA onto. In the G1 phase, catalytically inactive double hexamers of MCM2-7 are loaded onto double-stranded DNA (dsDNA) at replication origins1. In the S phase, MCM2-7 complexes are activated by the binding of CDC45 and GINS2 to form 11-subunit CMG complexes (CDC45, MCM2-7, GINS). Each CMG initiates DNA unwinding in opposite directions, forming the core unit that the replisome arranges itself around3.
Two decades ago the CMG helicase was first identified as an 11-subunit complex, essential for DNA replication4. Since then, our understanding of CMG has advanced considerably, from loading and activation5,6, to DNA unwinding and termination7. Traditional biochemical and structural biology techniques have been critical to many of these discoveries; however, these methods were often limited in their ability to study the more dynamic aspects of CMG. Single-molecule methods use physical manipulation of individual biomolecules to measure or visualize their activity one molecule at a time. This can be used to provide insight into the real-time dynamics of proteins that are often missed or undetectable by other techniques8,9.
Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay to visualize DNA unwinding by CMG helicase in real time. Purified, fluorescently labeled CMG is loaded onto the free 3&#39; end of long DNA containing a pre-made DNA fork structure. Linear DNA is stretched on a biotin-PEG coverslip in a microfluidic flow cell by tethering each end of the DNA sequentially to the surface. This approach allows for more uniform DNA tethering, which significantly reduces the variation that must be accounted for during data analysis. In the presence of ATP-&#947;-s, CMG is loaded onto the single-stranded DNA at the 3&#39; end of the fork. ATP-&#947;-s is a slowly hydrolyzable ATP analog, which permits CMG binding onto DNA but not unwinding. Subsequent addition of ATP, along with purified, fluorescently tagged RPA, activates CMG and initiates extensive DNA unwinding. Visually, CMG translocates along the DNA, leaving a growing tract of RPA-bound ssDNA behind it. The untethered DNA end travels with CMG, forming a &#34;tight ball&#34; due to compaction caused by RPA binding. The flow cell design allows the buffer to be exchanged at any point during unwinding, giving great control during and over each experiment.
This protocol is divided into four methods, which can be performed independently of one another. Section 1 describes the preparation of a 20-kb linear forked DNA substrate for single-molecule assays. Section 2 outlines the purification and fluorescent labeling of Drosophila melanogaster CMG (DmCMG). Key information about the expression of DmCMG is included in the notes section. Section 3 covers the preparation of a microfluidic flow cell that can be used on a TIRF microscope. Section 4 describes how to carry out the single-molecule DNA unwinding assay.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Single Molecule Total Internal Reflection Fluorescence Assay for Visualizing CMG-Mediated DNA Unwinding

single-molecule-total-internal-reflection-fluorescence-assay-for

Research

JoVE Journal

Biochemistry

167 Views. Single Molecule Total Internal Reflection Fluorescence Assay for Visualizing CMG-Mediated DNA Unwinding

Video: Single Molecule Total Internal Reflection Fluorescence Assay for Visualizing CMG-Mediated DNA Unwinding

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...