Stressed? Break-induced replication comes to the rescue!

Break-induced replication (BIR) is a homologous recombination (HR) pathway that repairs one-ended DNA double-strand breaks (DSBs), which can result from replication fork collapse, telomere erosion, and other events. Eukaryotic BIR has been mainly investigated in yeast, where it is initiated by invasion of the broken DNA end into a homologous sequence, followed by extensive replication synthesis proceeding to the chromosome end. Multiple recent studies have described BIR in mammalian cells, the properties of which show many similarities to yeast BIR. While HR is considered as "error-free" mechanism, BIR is highly mutagenic and frequently leads to chromosomal rearrangements-genetic instabilities known to promote human disease. In addition, it is now recognized that BIR is highly stimulated by replication stress (RS), including RS constantly present in cancer cells, implicating BIR as a contributor to cancer genesis and progression. Here, we discuss the past and current findings related to the mechanism of BIR, the association of BIR with replication stress, and the destabilizing effects of BIR on the eukaryotic genome. Finally, we consider the potential for exploiting the BIR machinery to develop anti-cancer therapeutics.

Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells.

Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50 bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells.

Tracking break-induced replication shows that it stalls at roadblocks.

Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA 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 diseases1,2. Previous studies have defined the enzymes that are required for BIR1-5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, 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. Interstitial telomeric DNA disrupts and terminates BIR progression, and 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 how BIR contributes to genome instability.