Fork Speed Analysis
Fork Speed Analysis - Related Videos
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...
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...
01:02
The DNA Replication Fork
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2023
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...
07:27
Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
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Cited by 10 •
2010
The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision.
13:31
High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis
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Cited by 42 •
2015
Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.
FAQs
The method translates the distance traveled by a replication fork into a speed measurement by relating DNA-track length to the defined labeling interval. Shorter tracks indicate slower progression, while missing or interrupted patterns can indicate stalling. Comparing these measurements across cellular conditions shows how replication dynamics change when genome duplication encounters stress or damage.
Sequential pulses create distinguishable labels in newly synthesized DNA, preserving information about the order and extent of synthesis. After the DNA fibers are stretched and imaged, the labeled tracks show how far forks progressed during each interval. This timing information helps identify unequal movement between forks and defects in continued replication.
Track patterns can distinguish several aspects of replication behavior, including fork slowing, stalling, asymmetry, and failure to restart. Asymmetry indicates that paired replication forks do not progress equivalently, whereas restart defects show that replication does not resume normally after interruption. These patterns provide a more detailed picture of genome stability than a single average speed.
Cells first receive sequential pulses of modified nucleotide analogs so newly synthesized DNA becomes labeled over defined intervals. DNA is then prepared as stretched fibers, the labels are detected by imaging, and the resulting track lengths are measured. Fork movement is calculated from each track length and its corresponding labeling interval, enabling comparisons among experimental conditions.
Researchers can compare fork progression under baseline and stressful cellular conditions, then determine whether stress produces slower movement, stalled forks, asymmetric progression, or impaired restart. These outcomes identify how strongly replication is disrupted and which aspect of fork behavior changes. The approach therefore connects altered cellular conditions with specific replication defects rather than treating stress as a single outcome.
Mutations, DNA damage, and replication-targeting drugs can be evaluated by measuring their effects on fork behavior. Changes in speed or track patterns indicate how these factors interfere with chromosome duplication. Because the assay examines replication directly on DNA fibers, it helps relate molecular or cellular perturbations to replication instability and potential defects in genome maintenance.