Related Experiment Video

Updated: Aug 9, 2025

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
07:27

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Published on: April 29, 2010

13.7K

Rolling Circle Replication and Bypass of Damaged Nucleotides

Josefin M E Forslund1, Gorazd Stojkovič1, Sjoerd Wanrooij2

  • 1Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

Methods in Molecular Biology (Clifton, N.J.)
|February 22, 2023
PubMed

Insights

Mitochondrial DNA (mtDNA) replication is vital for cell energy production. This study details a method to investigate how mtDNA replication handles DNA damage, crucial for understanding and potentially treating related diseases.

Area of Science:

  • Mitochondrial biology
  • Molecular genetics
  • DNA replication and repair

Background:

  • Faithful mitochondrial DNA (mtDNA) replication is essential for cellular energy production via oxidative phosphorylation.
  • Replication stalling due to DNA damage disrupts mtDNA maintenance, potentially leading to disease.
  • Understanding how the mtDNA replisome interacts with damaged DNA is critical.

Purpose of the Study:

  • To provide a detailed protocol for studying DNA damage bypass during mtDNA replication.
  • To establish an in vitro system for investigating mtDNA maintenance mechanisms.
  • To adapt assays for examining various aspects of mtDNA repair and replication fidelity.

Main Methods:

  • Utilizing a reconstituted in vitro mtDNA replication system.
  • Employing a rolling circle replication assay to study DNA damage bypass.
  • Using purified recombinant proteins to mimic cellular replication machinery.

Main Results:

  • The developed rolling circle replication assay allows for the investigation of DNA damage bypass.
  • The system can be adapted to study how the mtDNA replisome handles various types of DNA damage (e.g., oxidative, UV).
  • This reconstituted system provides a versatile platform for exploring mtDNA maintenance.

Conclusions:

  • The rolling circle replication assay is an effective method for studying mtDNA replication fidelity in the presence of DNA damage.
  • This in vitro approach facilitates detailed mechanistic studies of mtDNA maintenance.
  • The protocol can be adapted to explore diverse challenges faced by the mtDNA replisome, aiding in disease research.
Keywords:
DNA damageDNA polymeraseDNA replicationMitochondrial DNARolling circle

More Related Videos

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae07:55

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

1.9K
Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis10:36

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Published on: December 21, 2010

10.8K

Related Concept Videos

Homologous Recombination02:31

Homologous 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...
50.8K
Restarting Stalled Replication Forks02:37

Restarting 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,...
5.9K
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
10.1K
The DNA Replication Fork01:02

The 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...
36.3K
DNA Damage can Stall the Cell Cycle02:37

DNA Damage can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
9.2K
Genome Copying Errors02:46

Genome Copying Errors

DNA replication is a well-evolved process that copies millions of base pairs with high fidelity during each cell division. Occasionally a wrong base or a long stretch of wrong bases may get added to the daughter strands. If the errors are left unchecked, cells might accumulate several mutations that might endanger their  survival. Therefore, the copying errors are checked and repaired at three levels.
4.3K