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Related Experiment Video

Updated: Feb 6, 2026

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 Sugawara3,4

  • 1Cancer Program, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142.

Proceedings of the National Academy of Sciences of the United States of America
|February 4, 2026
PubMed
Summary

DNA double-strand break repair in yeast is mutagenic, favoring specific mutations like deletions over duplications. Microhomology-mediated template switching, crucial for repair, requires extensive homeology alignment for accuracy.

Keywords:
DNA repairmicrohomologymutationtemplate switchingyeast

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Area of Science:

  • Molecular Biology
  • Genetics
  • DNA Repair Mechanisms

Background:

  • DNA double-strand break (DSB) repair is a critical cellular process.
  • DSB repair pathways can be highly mutagenic, introducing genetic variations.
  • Understanding these mechanisms is key to comprehending genome stability.

Purpose of the Study:

  • To investigate the mutagenic outcomes of DSB repair in budding yeast.
  • To elucidate the structural and mechanistic features of the repair replication fork.
  • To characterize the role of microhomology and homeology in specific mutation types.

Main Methods:

  • Induction of DSBs using HO endonuclease in budding yeast.
  • Analysis of mutations arising from repair using an ectopic heterochromatic donor sequence (HMR::Kl-URA3).
  • Characterization of mutation types including base-pair substitutions, indels, intragenic deletions (IDs), tandem duplications (TDs), and interchromosomal template switches (ICTS).

Main Results:

  • DSB repair yielded 50% base-pair substitutions and 30% 1-bp indels, with a strong bias for deletions (-1) over insertions (+1) in homonucleotide runs.
  • Intragenic deletions (IDs) were 12 times more frequent than tandem duplications (TDs), suggesting a specific D-loop structure during repair.
  • ~10% of mutations were interchromosomal template switches (ICTS), occurring between homeologous sequences.
  • ICTS events require extensive adjacent homeology pairing, unlike IDs where microhomology is sufficient.

Conclusions:

  • The repair replication fork has an open, migrating D-loop structure that influences mutation patterns.
  • Microhomology-mediated repair processes, particularly ICTS, are significantly influenced by homeology alignment.
  • These findings refine our understanding of structural variant formation during DNA repair.