This paper demonstrates that the mechanism of break-induced replication (BIR) is significantly different from S-phase replication, as it proceeds via a migrating bubble driven by Pif1 helicase, results in conservative inheritance of newly synthesized DNA, and is inherently mutagenic. When DNA is repaired by homologous recombination, DNA synthesis is involved in the latter stages. Two papers published in this issue of Nature now define a role for the DNA helicase Pif1 in this reaction. They show that although the initial stages of break-induced replication (BIR) can occur normally in the absence of Pif1, synthesis from a migrating D-loop intermediate is compromised. The mechanism of replication during BIR involves a unique bubble-like replication fork that results in conservative inheritance of the new genetic material, in contrast to the S-phase replication that duplicates the genome before cell division, and is inherently mutagenic. The repair of chromosomal double strand breaks (DSBs) is crucial for the maintenance of genomic integrity. However, the repair of DSBs can also destabilize the genome by causing mutations and chromosomal rearrangements, the driving forces for carcinogenesis and hereditary diseases. Break-induced replication (BIR) is one of the DSB repair pathways that is highly prone to genetic instability1,2,3. BIR proceeds by invasion of one broken end into a homologous DNA sequence followed by replication that can copy hundreds of kilobases of DNA from a donor molecule all the way through its telomere4,5. The resulting repaired chromosome comes at a great cost to the cell, as BIR promotes mutagenesis, loss of heterozygosity, translocations, and copy number variations, all hallmarks of carcinogenesis4,5,6,7,8,9. BIR uses most known replication proteins to copy large portions of DNA, similar to S-phase replication10,11. It has therefore been suggested that BIR proceeds by semiconservative replication; however, the model of a bona fide, stable replication fork contradicts the known instabilities associated with BIR such as a 1,000-fold increase in mutation rate compared to normal replication9. Here we demonstrate that in budding yeast the mechanism of replication during BIR is significantly different from S-phase replication, as it proceeds via an unusual bubble-like replication fork that results in conservative inheritance of the new genetic material. We provide evidence that this atypical mode of DNA replication, dependent on Pif1 helicase, is responsible for the marked increase in BIR-associated mutations. We propose that the BIR mode of synthesis presents a powerful mechanism that can initiate bursts of genetic instability in eukaryotes, including humans.

Inverted DNA repeats are an at-risk motif for genetic instability that can induce both deletions and recombination in yeast. We investigated the role of the length of inverted repeats and size of the DNA separating the repeats for deletion and recombination. Stimulation of both deletion and recombination was directly related to the size of inverted repeats and inversely related to the size of intervening spacers. A perfect palindrome, formed by two 1.0-kb URA3-inverted repeats, increased intra- and interchromosomal recombination in the adjacent region 2,400-fold and 17,000-fold, respectively. The presence of a strong origin of replication in the spacer reduced both rates of deletion and recombination. These results support a model in which the stimulation of deletion and recombination by inverted repeats is initiated by a secondary structure formed between single-stranded DNA of inverted repeats during replication.

Expansion of triplex‐forming GAA/TTC repeats in the first intron of FXN gene results in Friedreich's ataxia. Besides FXN , there are a number of other polymorphic GAA/TTC loci in the human genome where the size variations thus far have been considered to be a neutral event. Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double‐strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin, which correlates with their propensity to adopt triplex structure and to block replication progression. We show that fragility is mediated by mismatch repair machinery and requires the MutSβ and endonuclease activity of MutLα. We suggest that the mechanism of GAA/TTC‐induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts.

Chromosomes of many eukaryotic organisms including humans contain a large number of repetitive sequences. Several types of commonly present DNA repeats have the capacity to adopt hairpin and cruciform secondary structures. Inverted repeats, AT- and GC-rich micro- and minisatellites, comprising this class of sequence motifs, are frequently found in chromosomal regions that are prone for gross rearrangements in somatic and germ cells. Recent studies in yeast and mammals indicate that a double-strand break occurring at the sites of unstable repeats can be an initial event in the generation of chromosome rearrangements. The repeat-induced chromosomal instability is responsible for a number of human diseases and has been implicated in carcinogenesis. In this review, we discuss the molecular mechanisms by which hairpins and cruciforms can trigger chromosomal fragility and subsequent aberrations in eukaryotic cells. We also address the relationship between secondary structure-mediated genetic instability and human pathology.

Break-induced replication (BIR) is a DNA double-strand break repair pathway that leads to genomic instabilities similar to those observed in cancer. BIR proceeds by a migrating bubble where asynchrony between leading and lagging strand synthesis leads to accumulation of long single-stranded DNA (ssDNA). It remains unknown how this ssDNA is prevented from unscheduled pairing with the template, which can lead to genomic instability. Here, we propose that uncontrolled Rad51 binding to this ssDNA promotes formation of toxic joint molecules that are counteracted by Srs2. First, Srs2 dislodges Rad51 from ssDNA preventing promiscuous strand invasions. Second, it dismantles toxic intermediates that have already formed. Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules. Overall, we uncover a new feature of BIR and propose that tight control of ssDNA accumulated during this process is essential to prevent its channeling into toxic structures threatening cell viability.