The major pathways of DNA double strand break (DSB) repair have key roles in suppressing genomic instability. However, if deployed in an inappropriate cellular context, these same repair functions can mediate chromosome rearrangements that underlie various human diseases, ranging from developmental disorders to cancer. Two major mechanisms of DSB repair predominate in mammalian cells, namely homologous recombination and non-homologous end joining. In this Review, we outline a 'decision tree' of DSB repair pathway choice in somatic mammalian cells, and consider how DSB repair dysfunction can lead to genomic instability. Stalled or broken replication forks present a distinctive challenge to the DSB repair system. Emerging evidence suggests that the 'rules' governing stalled fork repair pathway choice differ from those that operate at a conventional DSB.

The repair of chromosomal double strand breaks (DSBs) is crucial in 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 instability1–3. BIR proceeds by invasion of one broken end into a homologous DNA sequence followed by replication that can copy hundreds of kilobasepairs 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 carcinogenesis4–9. BIR employs the majority of known replication proteins to copy large portions of DNA, similar to S-phase replication10,11. It has thus 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 1000-fold increase in mutation rate compared to normal replication9. Here we demonstrate that 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 the evidence that this atypical mode of DNA replication, dependent on Pif1 helicase, is responsible for the dramatic 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.

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.

Break-induced replication (BIR) repairs one-ended double strand breaks (DSBs) similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human disease 1 , 2 . Previous studies have defined the enzymes required for BIR 1 – 5 ; however, understanding of initial and extended BIR synthesis as well as how the migrating D-loop proceeds through known replication roadblocks has been precluded by technical limitations. Here, using a newly developed assay, we demonstrate that BIR synthesis initiates soon after strand invasion and proceeds slower than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but fails to proceed beyond 30 kb, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32 but does not proceed efficiently. We demonstrate that interstitial telomeric DNA disrupts and terminates BIR progression. Also, BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and on how BIR contributes to genome instability.