Related Experiment Video
Updated: Dec 10, 2025
06:25
Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence
Published on: February 10, 2023
2.3K
Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication
Rujuta Yashodhan Gadgil1, Eric J Romer1, Caitlin C Goodman1
1Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA.
The Journal of Biological Chemistry
|September 3, 2020
Summary
Microsatellites in DNA can form non-B DNA structures, leading to replication-dependent DNA double-strand breaks (DSBs). These DSBs contribute to genome instability in diseases and cancers.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- DNA Repair Mechanisms
Background:
- Microsatellites are repetitive DNA sequences prone to instability and DNA double-strand breaks (DSBs).
- Replication-dependent DSBs at microsatellites are implicated in human developmental diseases and cancers.
- The formation of non-B DNA structures by microsatellites contributes to their instability.
Purpose of the Study:
- To investigate the causes and consequences of microsatellite DSBs using a novel reporter system.
- To analyze DSBs at expanded (CTG/CAG) and polypurine/polypyrimidine (Pu/Py) repeat structures.
- To understand the role of replication stress in microsatellite instability.
Main Methods:
- Designed a dual-fluorescence reporter system to detect DSBs at specific microsatellite sequences.
- Integrated reporter constructs with (CTG/CAG)100 and Pu/Py repeats and the c-myc replication origin.
- Utilized flow cytometry to detect reporter gene deletion following DSB repair.
- Manipulated DNA damage response protein levels (e.g., Rad18, Pol η, Pol κ, Mus81) via knockdown.
Main Results:
- Endogenous and exogenous replication stressors induce DSBs at (CTG/CAG)100 and Pu/Py microsatellites.
- DSBs are localized to the downstream edge of the (CTG)100 lagging-strand template.
- Microsatellite fragility depends on repeat length for (CTG/CAG) and replication polarity for (Pu/Py) repeats.
- Restriction-generated and replication-dependent DSBs are repaired via distinct mechanisms.
- Modulating DNA damage response proteins alters microsatellite sensitivity to replication stress.
- Replication stress and DSBs at microsatellites lead to break-induced replication and high-frequency mutagenesis.
Conclusions:
- Non-B DNA structure-forming microsatellites are susceptible to replication-dependent DSBs.
- These DSBs are a significant source of genome instability.
- Understanding microsatellite DSB mechanisms is crucial for addressing associated diseases.
Keywords:
DNA repairDNA replicationbreak-induced replicationcancergenomic instabilitymicrosatellite instabilitymutagenesismyotonic dystrophypolycystic kidney diseasetrinucleotide repeat diseaseMore Related Videos
Related Concept Videos
02:04Fixing Double-strand Breaks
14.0K
The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
14.0K
02:04Fixing Double-strand Breaks
4.1K
4.1K
02:31Homologous Recombination
61.6K
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...
61.6K
01:02The DNA Replication Fork
39.7K
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...
39.7K
02:37Restarting Stalled Replication Forks
6.1K
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,...
6.1K
01:03Single-Strand DNA Binding Proteins
16.3K
For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
16.3K