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Related Concept Videos

Fixing Double-strand Breaks02:04

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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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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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Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
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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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Related Experiment Video

Updated: May 22, 2025

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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Mutations and structural variants arising during double-strand break repair.

Simona Dalin1,2, Sophie Webster1,2, Neal Sugawara3

  • 1Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Biorxiv : the Preprint Server for Biology
|March 17, 2025
PubMed
Summary

Double-strand break (DSB) repair in yeast favors deletions over insertions, unlike normal replication. Microhomology-mediated repair generates specific structural variants like intragenic deletions and template switches.

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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
  • Genetics
  • DNA Repair Mechanisms

Background:

  • Double-strand break (DSB) repair pathways are critical for maintaining genomic integrity.
  • DSB repair can be mutagenic, leading to various genetic alterations.
  • Understanding the mechanisms of DSB repair is essential for comprehending genome evolution and disease.

Purpose of the Study:

  • To investigate the mutagenic outcomes of DSB repair in budding yeast.
  • To characterize the types and frequencies of mutations arising during repair.
  • To elucidate the role of microhomology and homeology in DSB repair-associated mutations.

Main Methods:

  • Induction of DSBs at the MAT locus in budding yeast using HO endonuclease.
  • Repair of DSBs using a transcriptionally silent HMR::Kl-URA3 donor sequence.
  • Analysis of mutation types, including deletions, insertions, and template switches, using DNA sequencing.

Main Results:

  • DSB repair favors -1 deletions over +1 insertions in homonucleotide runs, contrasting with replication.
  • Microhomology-bounded intragenic deletions (IDs) are 12 times more frequent than tandem duplications (TDs).
  • Interchromosomal template switches (ICTS) occur at regions of short microhomology and require extensive adjacent homeology alignment.

Conclusions:

  • DSB repair involves distinct mechanisms leading to specific mutation signatures.
  • Microhomology plays a crucial role in generating structural variants during repair.
  • Extensive homeology alignment is critical for interchromosomal template switching during DSB repair.