Comprehensive analysis of cis- and trans-acting factors affecting ectopic Break-Induced Replication

Abstract

Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating ectopic BIR efficiency, we found that ectopic BIR efficiency is the highest close to chromosome ends. The variations of ectopic BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of ectopic BIR depends on the length of DNA already synthesized. Ectopic BIR is more susceptible to disruption during the synthesis of the first ~35–40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes ectopic BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.

Author summary

DNA is a long molecule composed of two anti-parallel strands that can undergo breaks that need to be efficiently repaired to ensure genomic stability, hence preventing genetic diseases such as cancer. Homologous recombination is a major DNA repair pathway that copies DNA from intact homologous templates to seal DNA double strand breaks. Short DNA repair tracts are favored when homologous sequences for the two extremities of the broken molecule are present. However, when homologous sequences are present for only one extremity of the broken molecule, DNA repair synthesis can proceed up to the end of the chromosome, the telomere. This notably occurs at eroded telomeres when telomerase, the enzyme normally responsible for telomere elongation, is inactive, and at broken DNA replication intermediates. However, this Break-Induced Replication or BIR pathway is highly mutagenic. By initiating BIR at various distances from the telomere, we found that the length of DNA to synthesize significantly reduces BIR efficiency. Interestingly, our findings support two DNA synthesis phases, the first one being much less processive than the second one. Ultimately, this tends to restrain the use of this last resort DNA repair pathway to chromosome extremities notably when it takes place between non-allelic homologous sequences.