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Homologous Recombination02:31

Homologous Recombination

52.6K
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...
52.6K
Mismatch Repair01:20

Mismatch Repair

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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.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
5.2K
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

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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...
12.9K
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

10.2K
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...
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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

6.1K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
6.1K
Proofreading01:31

Proofreading

6.7K
Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase...
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Related Experiment Video

Updated: Sep 17, 2025

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
11:08

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Published on: June 19, 2018

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Pol θ-mediated end-joining uses microhomologies containing mismatches.

Yuzhen Li1, Ngoc K Dang1, Wei He1

  • 1Department of Epigenetics and Molecular Carcinogenesis, MD Anderson Cancer Center, Houston, TX, USA.

Nature Communications
|July 2, 2025
PubMed
Summary

DNA polymerase theta (Pol θ) repairs DNA by using short microhomologies. This study reveals Pol θ prefers mismatched microhomologies, influencing cancer mutation analysis.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • DNA polymerase theta (Pol θ) is crucial for repairing DNA double-strand breaks.
  • Understanding Pol θ's mechanism is vital as some cancers depend on it for survival.
  • Microhomology selection by Pol θ influences genomic stability and mutation patterns.

Purpose of the Study:

  • To investigate the microhomology selection process used by purified human Pol θ during DNA end-joining.
  • To determine the factors influencing Pol θ's choice of microhomologies.
  • To refine the definition of microhomology in the context of Pol θ activity.

Main Methods:

  • Utilized purified human Pol θ for in vitro end-joining assays.
  • Employed DNA sequencing to analyze products generated from diverse oligonucleotide libraries.
  • Investigated the role of sequence features near the 3' end in microhomology selection.

Main Results:

  • Pol θ preferentially selects short, internal microhomologies within 15 nucleotides of the single-stranded DNA terminus.
  • Selected microhomologies are frequently interrupted by mismatches.
  • Base pairing within 6 nucleotides of the 3' end significantly impacts microhomology choice.
  • Bidirectional synthesis is not required for Pol θ-mediated end-joining initiation.

Conclusions:

  • Pol θ's preference for mismatched microhomologies necessitates a revised definition of microhomology.
  • Findings advance the understanding of DNA repair mechanisms and Pol θ's role in genomic integrity.
  • This research has implications for analyzing cancer genomes and developing targeted therapies.