Related Experiment Video
Updated: Aug 6, 2026
07:18
Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique
Published on: October 27, 2011
Analyzing DNA Replication Fork Stability and Collapse Using Chromatin Fiber Analysis and the R-ODD-BLOBS Program
Kerenza Cheng1, Kazeera Aliar2, Roozbeh Manshaei3
1Molecular Science Graduate Program, Yeates School of Graduate and Postdoctoral Studies, Toronto Metropolitan University, Toronto, ON M5B 2K3, Canada.
Computational and Structural Biotechnology Journal
|July 23, 2026
Summary
We developed R-ODD-BLOBS to analyze DNA replication fork anatomy in fission yeast. This tool reveals how DNA repair proteins like Rad51 and damage markers like H2A.X localize around replication forks, especially after checkpoint loss.
Area of Science:
- Molecular Biology
- Genetics
- Computational Biology
Background:
- Understanding DNA replication fork dynamics is crucial for genome stability.
- Existing methods for analyzing replication fork structures are limited.
- Fission yeast (Schizosaccharomyces pombe) serves as a model organism for studying DNA replication and repair.
Purpose of the Study:
- To describe the anatomy of DNA replication forks.
- To analyze the localization of DNA replication, damage, and repair proteins relative to synthesized DNA.
- To investigate the impact of DNA replication checkpoint loss on fork structures.
Main Methods:
- Detection of replication (Cdc45, RPA), damage (H2A.X), and repair (Rad51) proteins on 5'-bromodeoxyuridine (BrdU)-labeled chromatin fibers.
- Analysis of chromatin fiber data using a novel computational tool, R-ODD-BLOBS (One Dimensional Data Boolean Logic Operations Binning System).
- Comparison of wild-type fission yeast with DNA replication checkpoint mutants (mrc1Δ and cds1Δ) after hydroxyurea treatment.
Main Results:
- R-ODD-BLOBS enables robust analysis of BrdU tract lengths and protein distributions.
- Distinct, checkpoint-dependent localization patterns of Rad51, Cdc45, RPA, and H2A.X were observed around replication forks.
- Increased Rad51 at forks in mrc1Δ suggests enhanced homologous recombination repair; Cdc45 enrichment in cds1Δ indicates helicase detachment; asymmetric H2A.X distribution suggests a barrier to damage signal extension.
Conclusions:
- R-ODD-BLOBS is a rigorous computational tool for analyzing large chromatin spread datasets.
- The study reveals detailed patterns of DNA replication length and protein components at replication forks.
- Loss of replication checkpoint function alters replication fork anatomy, impacting DNA repair pathway usage and fork stability.
More Related Videos
Related Concept Videos
02:37Restarting Stalled Replication Forks
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...
02:37Restarting Stalled Replication Forks
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...
01:02The DNA Replication Fork
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...
01:59Lagging Strand Synthesis
During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
01:29Replication in Eukaryotes
In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
Many Proteins Orchestrate Replication at the Origin
Eukaryotic replication follows many of the same...
Many Proteins Orchestrate Replication at the Origin
Eukaryotic replication follows many of the same...
02:31Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...