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Related Concept Videos

Homologous Recombination02:31

Homologous Recombination

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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...
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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...
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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...
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Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

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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...
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Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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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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DNA Helicases00:55

DNA Helicases

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DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
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Related Experiment Video

Updated: May 20, 2025

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

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Structural basis for Polθ-helicase DNA binding and microhomology-mediated end-joining.

Fumiaki Ito1,2,3, Ziyuan Li1, Leonid Minakhin4

  • 1Molecular and Computational Biology, Department of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA, 90089, USA.

Nature Communications
|April 19, 2025
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DNA polymerase theta (Polθ) is crucial for repairing DNA double-strand breaks (DSBs) in HR-deficient cancers. This study reveals Polθ

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Visualization of DNA Repair Proteins Interaction by Immunofluorescence
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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

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Related Experiment Videos

Last Updated: May 20, 2025

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
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Visualization of DNA Repair Proteins Interaction by Immunofluorescence
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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

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Area of Science:

  • Molecular Biology
  • Genomics
  • Structural Biology

Background:

  • DNA double-strand breaks (DSBs) threaten genomic integrity and can lead to cancer.
  • Homologous recombination (HR) and non-homologous end joining (NHEJ) are primary DSB repair pathways.
  • DNA polymerase theta (Polθ) is essential for microhomology-mediated end joining (MMEJ) in HR-deficient cells, presenting a therapeutic target.

Purpose of the Study:

  • To elucidate the molecular mechanisms of Polθ-mediated microhomology-mediated end joining (MMEJ).
  • To provide structural insights into the Polθ helicase domain's function in DSB repair.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) was used to determine structures of the Polθ helicase domain (Polθ-hel).
  • Structures were obtained in complex with DNA substrates featuring 3'-single-stranded DNA (ssDNA) overhangs.

Main Results:

  • Cryo-EM structures reveal sequential conformations of Polθ-hel during DNA binding, microhomology searching, and annealing.
  • Stepwise conformational changes in Polθ-hel subdomains and its dimeric state are critical for aligning 3'-ssDNA overhangs.
  • These structural dynamics facilitate microhomology search and annealing essential for MMEJ.

Conclusions:

  • The study identifies key molecular switches within Polθ-hel that regulate the MMEJ process.
  • These findings provide a structural basis for understanding Polθ-mediated DSB repair.
  • The research lays the foundation for developing targeted therapies against Polθ-hel in cancer treatment.