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Related Concept Videos

Overview of DNA Repair02:25

Overview of DNA Repair

33.9K
In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
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Overview of DNA Repair02:25

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Mismatch Repair01:36

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Mismatch Repair01:20

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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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Base Excision Repair01:54

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One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
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Long-patch Base Excision Repair01:02

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Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
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Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles
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Assaying Repair at DNA Nicks.

Luther Davis1, Yinbo Zhang1, Nancy Maizels1

  • 1University of Washington, Seattle, WA, United States.

Methods in Enzymology
|March 11, 2018
PubMed
Summary

DNA nicks, a common DNA damage, are efficiently repaired using specific DNA repair pathways. New reporter assays utilizing flow cytometry enable detailed analysis of these crucial repair mechanisms in human cells.

Keywords:
CRISPR/CasGene therapyGenome engineeringHomology-directed repairMutagenesisRecombination

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA nicks represent the most frequent type of DNA damage.
  • Recent findings highlight their role in initiating repairable damage.
  • Understanding DNA nick repair pathways is crucial for genome stability.

Purpose of the Study:

  • To detail reporter assays for analyzing DNA nick repair pathways in human cells.
  • To investigate the mechanisms of homology-directed repair and mutagenic end joining at targeted nicks.
  • To leverage flow cytometry for sensitive and high-throughput analysis of DNA repair.

Main Methods:

  • Development of site-specific nick-targeting enzymes.
  • Utilization of reporter systems for analyzing DNA repair outcomes.
  • Application of flow cytometry in 96-well plate format for assay convenience and sensitivity.
  • Employing depletion and ectopic expression to determine factor contributions.

Main Results:

  • Nicks are efficiently repaired via pathways involving single-stranded oligonucleotide donors.
  • These repair pathways are typically suppressed by RAD51.
  • Reporter assays facilitate the detailed analysis of repair pathway contributions.

Conclusions:

  • Reporter assays provide a sensitive and convenient method for studying DNA nick repair.
  • These assays enable mechanistic investigations into factors influencing repair pathways.
  • Understanding nick repair is vital for addressing DNA damage and maintaining genomic integrity.