RPA and Rad27 limit templated and inverted insertions at DNA breaks.

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• Formation of templated insertions at DNA double-strand breaks (DSBs) is frequently observed in cancer cells.
• The precise mechanisms and enzymatic players governing these templated insertions remain largely uncharacterized.

Purpose of the Study:

• To investigate the mechanisms underlying templated insertions at DSBs in yeast.
• To identify the enzymatic requirements and genomic features associated with templated insertion formation.

Main Methods:

• Utilized amplicon sequencing to analyze repaired loci in yeast following DSB induction.
• Investigated the role of key enzymes and DNA repair pathways, including DNA polymerase delta, nonhomologous end joining (NHEJ), alternative end joining (Alt-EJ), and RPA.
• Examined the impact of mutations affecting RPA levels/function and resection machinery (Sgs1, Exo1) on insertion formation.

Main Results:

• Identified very short (∼5-34 bp) templated inverted duplications at DSBs, formed via a foldback mechanism utilizing microhomologies.
• Demonstrated a hybrid enzymatic mechanism involving Polδ-mediated synthesis and either NHEJ or Alt-EJ.
• Observed increased templated insertions in mutants with deficient RPA or resection, including insertions from distant genomic locations and fragile sites.
• Found common complex insertions involving two sequences from the same locus in inverted orientation, suggesting microhomology-mediated template switching.

Conclusions:

• A shortage of RPA, a condition often seen in cancer cells, may promote the formation of templated insertions.
• The findings elucidate a novel mechanism for generating genomic instability through templated insertions at DSBs.
• Suggests that microhomology-mediated template switching is a key process in the formation of these insertions.