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Updated: Jul 11, 2025
Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
Published on: September 11, 2022
Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ-dependent mechanism
Amr M Al-Zain1,2, Mattie R Nester2, Iffat Ahmed2
1Program in Biological Sciences, Columbia University, New York, NY, 10027, USA.
Abstract:
Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease activity. Foldback priming at DNA double-strand breaks (DSBs) is one mechanism proposed for the generation of inverted duplications. However, the other pathway steps have not been fully elucidated. Here, we show that a DSB induced near natural inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity, but not Rad1 nuclease, trims the heterologous flaps formed after foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric inversion chromosome. Finally, we show that stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences or by deletion of one centromere.
Insights
DNA double-strand breaks near inverted repeats can cause foldback inversions, a common cancer rearrangement. DNA polymerase delta proofreading and Pol32 are key to this process, stabilizing resulting dicentric chromosomes.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Inverted duplications are frequent chromosome rearrangements in cancer.
- Foldback priming at DNA double-strand breaks (DSBs) is a proposed mechanism for their formation.
- The precise steps involved in generating inverted duplications remain unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms driving the formation of inverted duplications.
- To identify key proteins and enzymatic activities involved in this process.
- To understand how resulting dicentric chromosomes are stabilized.
Main Methods:
- Induction of DSBs near natural inverted repeats in yeast cells.
- Analysis of inverted duplication formation in cells deficient in Mre11, Sae2, and other DNA repair proteins.
- Assessment of DNA polymerase delta proofreading and Rad1 nuclease activity.
- Investigation of Pol32's role in the synthesis and stabilization pathways.
Main Results:
- DSBs near inverted repeats strongly promote inverted duplication in Sae2 and Mre11-deficient cells.
- DNA polymerase delta proofreading, not Rad1 nuclease, trims heterologous flaps.
- Pol32 is essential, indicating Pol δ-catalyzed fill-in synthesis of a hairpin-capped chromosome.
- Stabilization involves telomere capture or centromere deletion of the dicentric chromosome.
Conclusions:
- The study details a pathway for inverted duplication formation initiated by DSBs near repeats.
- DNA polymerase delta and Pol32 are critical for generating these rearrangements.
- Mechanisms for stabilizing the resulting dicentric chromosomes have been identified.
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