Extensive DNA end processing by exo1 and sgs1 inhibits break-induced replication

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA double-strand breaks (DSBs) are repaired via pathways like gene conversion and break-induced replication (BIR).
• BIR involves replication to the chromosome end, leading to extensive loss of heterozygosity (LOH).
• The role of DNA resection in BIR fidelity and frequency is not fully understood.

Purpose of the Study:

• To develop and validate a novel assay for studying break-induced replication (BIR) in Saccharomyces cerevisiae.
• To investigate the impact of DNA resection, specifically the 5'-3' resection pathway, on BIR frequency and fidelity.
• To analyze the consequences of impaired resection on the generation of chromosome fragments via BIR.

Main Methods:

• Development of a Saccharomyces cerevisiae BIR assay using a plasmid with a telomere seeding sequence and homology to chromosome III, separated by an I-SceI site.
• Induction of DSBs using the I-SceI endonuclease in vivo.
• Analysis of BIR product formation and DNA synthesis extension using physical methods in wild-type and mutant strains (exo1Δ, sgs1Δ, exo1Δ sgs1Δ).
• Comparison of BIR with a plasmid-chromosome gene conversion assay.

Main Results:

• The BIR assay successfully generated stable chromosome fragments (CFs) in Saccharomyces cerevisiae.
• The exo1Δ sgs1Δ mutant, deficient in 5'-3' resection, showed a 39-fold increase in BIR frequency compared to wild-type cells.
• While DNA synthesis extension was observed in multiple mutants, fully repaired BIR products were primarily visible in the exo1Δ sgs1Δ mutant.
• Reduced resection in the exo1Δ sgs1Δ mutant led to an increase in chromosome rearrangements due to BIR template switching, indicating decreased fidelity.

Conclusions:

• Inhibition of 5'-3' DNA resection significantly enhances the frequency of break-induced replication (BIR).
• Reduced resection, while promoting BIR, can compromise the fidelity of homologous recombination, leading to increased chromosomal instability.
• The developed BIR assay provides a valuable tool for studying the mechanisms and fidelity of this critical DNA repair pathway.