Break-induced replication occurs by conservative DNA synthesis

Abstract

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.

Keywords: Pol32; cell cycle; translocation.

Fig. 1.

Model for conservative or semiconservative DNA synthesis during BIR. If the Rad51-catalyzed D-loop migrates to the chromosome end (tel) and lagging-strand synthesis initiates on the displaced nascent strand, both newly synthesized strands (shown in blue) will segregate with the recipient (R) chromosome. Cleavage of the strand invasion intermediate by a structure-selective nuclease and establishment of a replication fork is predicted to result in semiconservative synthesis detected by segregation of the newly synthesized strands to donor (D) and recipient chromosomes.

Fig. 2.

BIR efficiency is determined by the length of DNA to be synthesized. (A) Schematic showing the locations of the recipient and donor cassettes on Ch V and XI, respectively, and the PCR primer pairs used to detect DSB formation by HO endonuclease (D1, D2), BIR products (P1, P2), and control locus (C1, C2). (B) BIR efficiency determined by CFU Lys⁺ YPGal/CFU YPGlu for each of the indicated strains from three independent trials; error bars show SD. (C) Schematic of the donor chromosomes used to measure BIR efficiency showing the distance from the donor homology to the telomere. The TRP1-ys2 cassette and primers used to detect BIR are the same for all of the donor chromosomes.

Fig. 3.

BIR product formation is delayed for the 70-kb donor strain compared with the 15-kb donor strain and occurs with highest efficiency in G₁ released cells. (A) FACS profiles and PCR to detect DSB (D primers) and BIR product (P primers) from cells of the indicated strains that were arrested in G₁ and released at the time of HO induction. (B) Kinetics of BIR product formation in G₁-released cells determined by the ratio of BIR to control (C) PCR products from three trials; error bars show the mean ± SD. (C) FACS profiles and PCR to detect DSB and BIR product from cells that were arrested in G₂-M at the time of HO induction. (D) Kinetics of BIR product formation in G₂-M arrested cells; error bars show the mean from three trials ± SD. (E) Schematic showing sizes of digestion products for the recipient chromosome before and after HO cutting, the donor chromosome and BIR product. The pale orange bar indicates the position of the hybridization probe and the vertical arrows show the location of EcoRV sites. (F) Southern blot of EcoRV-digested genomic DNA of the indicated strains before and after HO induction.

Fig. 4.

Newly synthesized DNA strands are associated with the recipient chromosome. (A) Detection of the BIR translocation product in G₁-released cells by PFGE and Southern blot hybridization using a probe specific to Ch XI. Lower panel shows the BIR product detected by PCR for the same samples. (B) Western blot of the pulsed field gel of the indicated strains 6 h after HO induction shows greater BrdU incorporation into the recipient chromosome compared with the donor chromosome. (C) Quantification of the relative BrdU signal (Ch V–VIII/Ch XI) from three independent trials; error bars show SD. (D) Schematic showing the locations of the recipient and donor cassettes on Ch V and I, respectively, and the PCR primer pair used to detect BIR products (P1, P2). BIR generates a novel-sized chromosome of 606 kb that can be resolved from the donor and recipient chromosomes by PFGE. (E) BrdU was added to the culture 2 h after HO induction and used to track incorporation into recipient or donor chromosomes by PFGE. The black arrows indicate the positions of the recipient chromosomes and white arrows mark the positions of the donor chromosomes.