Helicase Unwinding
Helicase Unwinding - Related Videos
07:37
Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase
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Cited by 1 •
2024
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.
00:55
DNA Helicases
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2020
DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
04:10
Helicase Activity Measurement of a Target Protein Using Biotin-Labeled RNA Duplexes
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2025
In this video, we demonstrate the procedure to determine the helicase activity of a target protein to unwind the biotin-labeled dsRNA substrate. The activity of the enzyme was identified by analyzing the electrophoretic mobility shift, followed by a chemiluminescence assay using chemiluminescent enzyme-conjugated streptavidin.
01:02
The DNA Replication Fork
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2020
An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork. Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in...
02:37
Restarting Stalled Replication Forks
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2020
DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...
FAQs
ATP hydrolysis supplies the energy that allows helicase to move along one nucleic acid strand. This movement proceeds directionally from a nucleic acid junction and progressively disrupts the hydrogen bonds holding the duplex together. Because the enzyme couples chemical energy to movement, strand separation can advance through the nucleic acid rather than occurring as a single, passive opening event.
A nucleic acid junction provides the point at which helicase engages the paired strands and begins its movement. Binding at this structural transition positions the enzyme to track along one strand while separating the duplex. This arrangement helps explain how helicase activity targets double-stranded or structured regions that must be opened before other nucleic acid processes can proceed.
Disrupting hydrogen bonds temporarily separates paired nucleic acid strands, exposing genetic information that is otherwise held within the duplex structure. This accessibility allows copying and gene expression processes to act on the nucleic acid. The same principle also supports recombination and repair, where structured or paired regions may need to be resolved before processing can continue.
During replication, helicase activity opens the paired nucleic acid ahead of DNA polymerases, while the polymerases copy the newly accessible information. Their coordination links strand separation with synthesis rather than treating these as independent events. This relationship is important because replication depends on continued access to the template as helicase progresses through the duplex.
Helicase activity resolves double-stranded or structured nucleic acid regions that can obstruct transcription, recombination, or repair. By making these regions accessible, it supports the molecular steps needed to read, rearrange, or restore genetic information. Its contribution extends beyond replication, showing that strand separation is a recurring requirement across several forms of nucleic acid metabolism.
Studying helicase unwinding helps researchers connect ATP use, directional movement, strand separation, and nucleic acid accessibility with genome stability. It also provides a way to investigate how cells manage nucleic acid metabolism across replication, transcription, recombination, and repair. When helicase defects or inhibitors alter these activities, the resulting effects can reveal disease mechanisms and possible therapeutic targets.