Mre11 and Ku regulation of double-strand break repair by gene conversion and break-induced replication

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair Mechanisms

Background:

• The Mre11-Rad50-Xrs2 (MRX) and Ku complexes are crucial for initiating DNA double-strand break (DSB) repair via homologous recombination (HR).
• Previous studies indicated that impaired single-strand resection, influenced by mre11 mutations, reduces gene conversion tract lengths and crossovers in plasmid-based assays.
• The role of end resection extent in regulating heteroduplex formation and tract lengths during chromosomal HR remains unclear.

Purpose of the Study:

• To investigate whether mre11 mutations affect gene conversion tract lengths and crossover frequencies during homologous recombination between chromosomes in diploid yeast.
• To determine if the extent of DNA end resection regulates heteroduplex formation and tract lengths in a chromosomal context.
• To elucidate the roles of Mre11 and Ku in different DNA repair pathways, including break-induced replication (BIR).

Main Methods:

• Utilized diploid yeast strains with specific mutations in Mre11 (mre11Δ, nuclease-defective mre11) and Ku (yku70Δ).
• Assessed gene conversion tract lengths and crossover frequencies during HR between homologous chromosomes.
• Employed plasmid-chromosome DSB repair and gap repair assays to compare tract lengths under different repair conditions.

Main Results:

• Mre11 mutations reduced HR efficiency but did not alter gene conversion tract lengths or crossovers on homologous chromosomes, despite reduced end resection.
• Increased end resection in yku70Δ mutants also failed to affect tract lengths, indicating resection extent does not regulate tract length in chromosomal HR.
• While HR outcome was largely unaffected, mre11 mutants showed increased break-induced replication (BIR) and chromosome loss, suggesting Mre11's role is limited to early HR stages.
• yku70Δ suppressed BIR in mre11 mutants, and Ku was found to function in Rad51-dependent BIR, whereas Mre11 functions in Rad51-independent BIR.

Conclusions:

• Heteroduplex formation and tract lengths during chromosomal DSB repair are not regulated by the extent of end resection.
• Mre11's function in mitotic HR is primarily in the early stages, and its role in BIR is independent of Rad51.
• Ku functions in Rad51-dependent BIR, highlighting distinct roles for MRX and Ku complexes in DNA repair pathway choice.