Break-induced replication (BIR) refers to recombination-dependent DNA synthesis initiated from one end of a DNA double-strand break and can extend for more than 100 kb. BIR initiates by Rad51-catalyzed strand invasion, but the mechanism for DNA synthesis is not known. Here, we used BrdU incorporation to track DNA synthesis during BIR and found that the newly synthesized strands segregate with the broken chromosome, indicative of a conservative mode of DNA synthesis. Furthermore, we show the frequency of BIR is reduced and product formation is progressively delayed when the donor is placed at an increasing distance from the telomere, consistent with replication by a migrating D-loop from the site of initiation to the telomere.H omologous recombination (HR) is an important mechanism to repair DNA double-strand breaks (DSBs) that occur spontaneously during cell growth or following exposure to DNA damaging agents (1). HR relies on the presence of a homologous duplex to template repair of the broken chromosome and is generally considered to be an error-free mechanism. However, HR can lead to a local loss of heterozygosity (LOH) if the recombining sequences are not identical, and to extensive LOH if repair is associated with a crossover between chromosome homologs. Furthermore, if a repeated sequence at an ectopic site is used as the sequence donor and recombination is associated with crossing over, translocations can occur (2, 3). When both ends of the DSB share homology with the donor duplex sequence, HR proceeds by a two-ended mechanism, such as DSB repair or synthesis-dependent strand-annealing (SDSA) (4-6). However, if coordination of the two ends is not maintained or only one end of the break is available, such as at a critically short telomere, repair can occur by break-induced replication (BIR) (7). In this case, following strand invasion replication occurs to the end of the chromosome to generate a stable repaired product (8,9). This process can cause very long gene conversion tracts and significant LOH, and nonreciprocal translocation if invasion occurs at a dispersed repeated sequence (10).The repair of DSBs by HR requires the 5′-3′ nucleolytic degradation of the DNA ends to form invasive 3′ single-stranded DNA (ssDNA) tails (1). The 3′ ssDNA tail created by end resection is bound by Rad51 to form a nucleoprotein filament that searches for homology and promotes pairing between the ssDNA bound by Rad51 and complementary sequence in the donor duplex forming a D-loop intermediate. The invading 3′ end is then used to prime DNA synthesis templated by the donor sequence. If the invading 3′-tail is displaced by helicases and anneals with the other end of break, repair by SDSA results in noncrossover products. If the second end of the break is captured by the D-loop, a double Holliday junction can be generated after DNA repair synthesis and ligation. Double Holliday junctions can either be dissolved by the Sgs1 helicase and Top3 topoisomerase to form noncrossover products or resolved by endonucleases to generate cr...

Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.

SUMMARY Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

SUMMARY DNA double strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular, break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Over-expression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.