Migrating bubble during break-induced replication drives conservative DNA synthesis

Abstract

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 instability. 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 telomere. 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 carcinogenesis. BIR uses most known replication proteins to copy large portions of DNA, similar to S-phase replication. 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 replication. 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.

Figure 1. The mode of DNA synthesis during BIR

a, The models of BIR. (i), Replication fork proceeds semiconservatively. (ii–iv), Migrating bubble leads to conservative inheritance of new DNA. Synchronous (ii) and asynchronous (iii,iv) synthesis of leading and lagging DNA strands. b, (i), The BIR frameshift mutation assay. A DSB is induced at MATa of the recipient chromosome (Chr) III. lys2 reporter is inserted in the donor chromosome 16 or 36 kb telomere-proximal from MATα-inc. Lys⁺ mutations would be inherited equally by the donor (D) or recipient (R) if BIR is semiconservative (ii), but only by recipient if BIR is conservative (iii).

Figure 2. BIR-induced mutations

a, The sequencing of the separated donor and recipient chromosomes of heterozygous Lys⁺ mutants. b, The effect of pif1Δ on BIR-induced frameshifts. Medians of mutation rates are shown. The arrows represent a reduction as compared to wt. c, The assay to study BIR-induced base substitutions in ura3–29 reporter. d, Depending on orientation, the selectable position of ura3–29 leading strand includes cytosine (C) or guanine (G). e, MMS amplifies BIR-induced base substitutions in orientation-dependent way. The arrows indicate an increase as compared to no-MMS control. See Extended Data Tables 1 and 2 for the details of statistical analysis and for the ranges of medians shown in e and b.

Figure 3. DNA synthesis during BIR is conservative

a, Experimental system to assay BIR using dynamic molecular combing including the position of hybridization probes P1, P2 and P3. b, (i), BrdU incorporation in the recipient is expected from conservative BIR (red). (ii), Formation of half-crossovers in pif1Δ leads to short patches of BrdU in recipient. c, Donor and recipient chromosomes separated using PFGE. d, The summary of molecular combing analysis. e, The donors and recipients of wt (PIF1) and pif1Δ. Each molecule was hybridized with P1, P2, P3 probes (green tracts) and treated with anti-BrdU antibodies (red tracts).

Figure 4. Molecular intermediates of BIR

a, D-loop migration during coordinated (i) and uncoordinated (ii, iii) leading- and lagging-strand synthesis. b. Schematic of 2D gel with BIR bubbles forming an arc (1,2) with an extension (3) representing ssDNA tail. Annealing with PstO3 and PstO4 allows PstI digestion changing the mobility of the intermediate (red, 2’). c, 2D analysis of Y-arc during normal replication (0Hr) and bubble-like structures at time points following BIR induction hybridized to LYS2-specific probe. d, High molecular-weight tails (arrows) disappear following annealing with PstO3 and PstO4. The arc is absent in no-cut controls. E, BIR intermediates highlighted with HPH-specific probe.