Mutations and structural variants arising during double-strand break repair.

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.