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Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA [1]

['Rebecca Martin', 'Department Of Genome Sciences', 'University Of Washington', 'Seattle', 'Washington', 'United States Of America', 'Claudia Y. Espinoza', 'Christopher R. L. Large', 'Joshua Rosswork', 'Cole Van Bruinisse']

Date: 2024-01

Inherited and germ-line de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA—origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres—suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at a replication fork, followed by integration via homologous recombination, generates inverted interstitial triplications.

Chromosomal rearrangements are a potent source of genetic variation in humans and other organisms. One specific type of rearrangement involves the increase in copies of segments of the genome. The variation in gene dosage that these rearrangements can cause has been associated with a wide range of neurological and other human disorders. A specific puzzling form of copy number increase consists of three tandem copies with the central copy in inverted orientation. How this rearrangement occurs is of great interest, yet the mechanisms responsible are only inferred by examining the sequence of final inverted products. Yeast provides a unique model system to explore the underlying molecular defects that give rise to inverted triplications. While the favored hypothesis suggests that double stranded DNA repair is the causative agent, we find that a particular form of template switching between strands at the replication fork, not a double stranded DNA break, is the initiating event. Using the awesome power of yeast genetics, we provide evidence in two different assays for this unique replication error that we call ODIRA (for Origin Dependent Inverted Repeat Amplification) and propose that it can also explain this form of copy number variant seen in human evolution and disease.

Funding: This project was supported by National Institutes of Health ( https://www.nih.gov ) grants R01 GM018926 and R35 GM122497 to BJB and MKR; and National Science Foundation ( https://www.nsf.gov ) grant 1120425 and National Institutes of Health ( https://www.nih.gov ) grant P41 GM103533 to MJD. AWM and CRLL were supported in part by National Institutes of Health ( https://www.nih.gov ) grant T32 HG00035. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To determine whether the hairpin ODIRA model could explain the occurrence of inverted triplication of the SUL1 locus in sulfate-limited chemostats [ 16 – 18 ], we sequenced 31 independent populations of cells that had been passaged for ~250 generations. By focusing on split reads that define junctions of inversions, we discovered that nearly a third of the cultures had different numbers of Cen-proximal and Tel-proximal inverted junctions, suggesting that there were inverted linear segments produced during the experiments. Moreover, the spacing of short-inverted repeats, the orientation of the inverted junctions and the positions of the junctions with respect to the replication map of this region of chromosome II provide further evidence that template switching between strands at a replication fork, not double strand DNA breakage, initiates inverted gene amplification in yeast.

Creation of identical linear extrachromosomal intermediates could be explained by repair of a double stranded DNA (dsDNA) break that forms intrastrand hairpins at short, interrupted palindromes near the break. To distinguish between these two mechanisms we carried out the selection for Ura+ clones under two conditions: (1) altering the strand dynamics at replication forks by reducing the availability of dNTPs with two different concentrations of hydroxyurea, and (2) increasing dsDNA breaks by targeting CRISPR/Cas9 centromere proximal to the SUL1 locus. These experiments suggest that template switching between the leading and lagging strands at a replication fork, but not double stranded DNA breaks, initiates the production of inverted amplicons.

To capture potential inverted extrachromosomal intermediates, we integrated a 5’ fragment of URA3 (“ura”) on chromosome II at the SUL1 locus, a region that is prone to inverted triplications in sulfate-limited cultures. On chromosome IX we integrated the overlapping partially complementary “ra3” fragment. Direct recombination between the homologous regions on chromosome II and IX would produce unstable dicentric chromosomes; however, an ODIRA event—a circular inverted dimeric ura fragment integrating into chromosome IX—would recreate a functional URA3 gene along with a partial inverted triplication on chromosome IX. Among the hundreds of clones analyzed we failed to detect any Ura+ clones that were consistent with integration of circular intermediates into chromosome IX. Instead, we identified Ura+ clones with genomic rearrangements that could be explained by a recombination between an inverted linear ura fragment from chromosome II with the ra3 fragment on chromosome IX (ODIRA hairpin model).

The nature of the inverted junctions inspired the ODIRA model. One well characterized example from the human literature seemed to fit perfectly with ODIRA because the triplication that occurred in the father of the female proband was a 2:1 mixture of SNPs from his two homologues [ 20 ]. An inverted dimeric circle that arose from one homologue and inserted into the other homologue during or preceding meiosis could be the explanation for the 2:1 SNP ratio. This case provided us with the stimulus to artificially recreate such an event in yeast by asking whether we could detect the movement of the inverted dimeric circle (produced by template switching across the replication fork) to a new location in the yeast genome. We designed a split-ura3 construct to identify the movement of potential extrachromosomal intermediates from one location to another by the recreation of a functional URA3 gene from two overlapping partial ura3 fragments.

Two other models of template switching—FoSTeS and MMBIR—have been proposed to account for complex chromosomal rearrangements involving distant interactions [ 9 , 10 ]. These models involve the migration of the 3’ end of a nascent strand from a replication fork or the exposed 3’end from a double stranded break to other regions of homology elsewhere in the genome. At the new site, replication is reestablished, generating a junction between two disparate regions of the genome. To explain complex rearrangements, the model proposes that the same strand makes multiple sequential invasion/extension attempts in a single cell cycle. Inverted triplications would not require long-distance template switching since the homologous template is the opposite strand at the replication fork ( Fig 1A .2 and 1B.1).

The amplicon can also arise in a two-step ODIRA mechanism ( Fig 1B .1; “hairpin” ODIRA) where the two template switches are temporally uncoupled. Ligation of leading and lagging strands at just the single fork moving toward the centromere (centromere-proximal junction; CJ) could produce an intermediate consisting of a hairpin capped linear segment extending to the telomere that could persist as an extrachromosomal inverted linear duplex after replication ( Fig 1B .2-3). (We use the term hairpin to refer specifically to the expelled double stranded linear with a single stranded loop at one end. After replication we refer to the completely double stranded product as an inverted linear molecule.) Recombination with the initiating chromosome does not result in integration, but rather just shuffles the telomeric arms between the inverted linear and the chromosome. However, cells containing the inverted linear would enjoy a selective advantage in the sulfate-limited chemostat. In a subsequent cell cycle, a second leading-to-lagging strand template switch in the fork moving toward the telomere (telomere-proximal junction; TJ; Fig 1B .4-6) of the inverted linear would generate a doubly inverted linear that could recombine with the SUL1 chromosome and generate the inverted triplication, improving its stability and selective advantage that leads to this clone sweeping the population. Notice that recombination with either of the internal repeats produces the triplication while recombination with either of the more distal repeats just shuffles the telomeres.

In the following diagrams thick lines indicate double stranded duplexes and thin lines indicate individual single strands. A) In our ODIRA model we propose that stalled forks (2a) provide an opportunity for a template switch between the nascent leading strand and the lagging strand template that occurs at short, interrupted inverted repeats (2b). Extension of the displaced leading strand and its ligation to an Okazaki fragment (2c) results in a covalent linkage between the leading and lagging nascent strands that can be expelled from the chromosome by an incoming fork from an adjacent origin (2d, and 3). A similar template switch at the divergent fork results in an extrachromosomal, self-complementary, single-stranded circular molecule (dogbone; 3). In the next cell cycle, the dogbone can replicate from its resident origin creating a duplex circular molecule that has two copies of the SUL1 region in inverted orientation (4). Recombination of the inverted dimeric circular molecule into the chromosome through homology with the SUL1 region creates a triplication with the center copy in inverted orientation (5). The inversion junctions (Cen-proximal and Tel-proximal; CJ and TJ, respectively) map to the genomic, short interrupted, inverted repeats where the template switching occurred. B) The Cen-proximal and Tel-proximal inversions can occur in different cell cycles, generating inverted linear molecules. After the second, telomere-proximal junction is created, the doubly inverted linear molecule can recombine with the SUL1 region creating an inverted triplication that is identical to that produced by the dogbone ODIRA model. The gray shaded panels in A and B show both strands of the DNA as thin lines to highlight the mechanism of template switches and the expelled transient intermediates (dogbones and hairpins). The dotted rectangles indicate the final chromosomal products of the two pathways.

During long-term growth of the common laboratory strains of haploid budding yeast Saccharomyces cerevisiae in chemostats limiting for sulfate, we routinely recover inverted triplications of the SUL1 locus, which encodes the primary sulfate transporter [ 16 , 17 ]. The reproducibility of this outcome provides an ideal system and opportunity to investigate the mechanism that gives rise to this form of gene amplification. Although the size of the amplified region varies, several structural features appear to be invariant ( Fig 1A .1): (1) the amplified segment contains at least one origin of replication, (2) the junctions that mark the boundaries of the amplified segment occur at pre-existing short, interrupted inverted repeats, and (3) the arms of the inverted repeats used for amplification are within a hundred base pairs of each other [ 16 – 18 ]. We have proposed a unique template-switching model, called ODIRA (Origin-Dependent Inverted Repeat Amplification; [ 19 ]), in which the leading strands at divergent, stalled replication forks become ligated to the Okazaki fragments on the lagging strands due to strand migration ( Fig 1A .2; “dogbone” ODIRA). Displacement and replication of the closed loop of newly synthesized DNA—the dogbone—gives rise to an inverted, dimeric, circular DNA molecule containing SUL1 and the adjacent origin of replication ( Fig 1A .3-4). (We use the term dogbone to refer specifically to the expelled closed DNA with single stranded loops. After replication we refer to the double stranded product as an inverted dimeric circular molecule.) Subsequent integration of the inverted dimeric circle into the chromosome at the original locus generates the triplication with an inverted center copy without disturbing the distal chromosomal sequences ( Fig 1A .5).

Copy number variation (CNV) refers to both increases and decreases in copies of genomic segments. In humans, many CNVs not only distinguish us from our close primate relatives, but some arise de novo and are associated with a range of human disorders [ 1 – 5 ]. One of the most common forms of CNV found in the human genome is the repetition of large genomic segments (referred to collectively as segmental duplications). Although the extra copies can be found as a direct repeat at the original locus, they may also be found at dispersed sites on the same or different chromosomes [ 6 ]. There are three major pathways that are thought to give rise to changes in copy number through distant interactions: non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ) and template switching during replication (FoSTeS and MMBIR) (Reviews: [ 7 – 13 ]). However, these mechanisms fail to easily and/or completely explain the formation of an unusual form of segmental duplication that involves a tandem triplication, where the central copy is inverted within an otherwise unrearranged chromosome. This form of CNV is seen in many human disorders and is likely under-reported because determining its inverted structure is technically challenging [ 8 , 14 , 15 ].

For an inverted split-read junction to be considered significant, we required that at least two independent chromosome fragments from the same culture produce the same junction sequence. Inverted junctions represented by a single read were considered to be PCR artifacts produced during the sequencing protocol, while those represented by more than one unique read were copies of in vivo generated inversions.

Split reads are defined as 150 bp reads that map to two non-contiguous regions of the yeast genome [ 17 ] and were manually curated on IGV (Integrative Genomics Viewer; [ 29 ]) as falling into one of five categories: (1) inverted junctions have reads divided between two sections of chromosome II on opposite DNA strands (n = 92); (2) direct repeat junctions have the two segments of the sequence from the same strand of chromosome II (n = 4;); (3) de novo telomere additions contain part of a chromosome II sequence adjacent to a C 1-3 A/G 1-3 T telomere sequence (n = 0); (4) telomere translocation junctions have the terminal SUL1 part of chromosome II-R attached to an existing telomere (n = 6); and (5) internal translocation junctions join chromosome II sequence to a repetitive element elsewhere in the genome—such as Ty elements, solo delta elements, tRNAs, and polyA/polyT or CAG stretches (unquantified). Inverted junction sequences (category 1) were catalogued ( S6 Table ) and characterized by the size of the inverted sequence at the discontinuity, the spacing between the two inverted sequences, and orientation of the inversion event. To establish baselines for all available inverted repeats we used the EMBOSS Palindrome program ( https://www.bioinformatics.nl/cgi-bin/emboss/palindrome ) to find all potential inverted repeats and their spacing across the terminal ~80 kb of Chromosome II with an interrupted inverted repeat structure of ≤250 bp using the sacCer3 version of the genome.

150 bp paired-end sequence of whole genome fragments purified from population samples on the last day of the chemostat run were prepared and analyzed as described [ 17 ]. Median genome read depth for the 31 sulfate-limited chemostats was ~125. Read depth analysis for the sulfate-limited cultures showed amplification of the SUL1 gene and flanking sequences while none of the 32 glucose-limited chemostats had amplifications in the SUL1 region. All sequencing data are available in the NIH Sequence Read Archive (SRA) under BioProject ID PRJNA1016460.

Note: Interstitial inverted triplications in the human CNV literature are referred to as a “triplicated segment embedded in an inverted orientation between two duplicated sequences (DUP-TRP/INV-DUP)” [ 27 ]. They are found associated with a variety of genetic syndromes but remain an underappreciated form of CNV, primarily because the inverted nature of the amplicon junctions poses a challenge for DNA sequencing platforms [ 15 ]. While arrayCGH has largely been replaced by long read sequencing in genomic research there are inherent problems with nanopore sequencing of inverted templates [ 28 ]. We suggest that a modified protocol of aCGH can easily detect inverted boundaries of amplified regions.

Genomic DNA from frozen chemostat samples from generation ~250 was isolated by the NIB-n-Grab protocol (see above), a modified version of the Smash-and-Grab protocol [ 25 ] that results in the recovery of DNA 20–50 kb in size. In this protocol, cells are broken by vortexing with glass beads in a buffer that stabilizes nuclei (NIB; [ 26 ]). aCGH was performed using Agilent 4x44k microarrays with probes spaced every 290 nt on average. Hybridization was executed as described previously [ 21 ]; however, sonication of the DNA samples was performed after in vitro labeling, rather than before labeling. This alteration allows inverted junctions to be identified by the gradual increase in signal—from single copy to multiple copies—at the site of inversion (see S2 Fig ). aCGH data for the relevant chromosomes are available in S4 and S5 Tables.

The split-ura3 strain was transformed with plasmid pYCpGal that include a yeast centromere, the yeast LEU2 gene, the GAL1 promoter driving Cas9 expression, and a guide RNA cloning site. The Cas9-guide cassette was derived from plasmid pML104 (Addgene) ( S1 Fig ). Guide RNAs expressed from this plasmid directed cutting to either position 708.260 kb (pYCpGAL-708b) or 792.883 kb (pYCpGAL-792b) on chromosome II ( S3 Table ). Relative to SUL1, the sites are centromere-proximal and centromere-distal, respectively. Standard LiAc transformation was used to introduce a no-guide plasmid, the 708 plasmid or the 792 plasmid into the split-ura3 strain, selecting for transformants on -leucine, +glucose plates. Single colonies were then used to inoculate 1 mL of –leucine +glucose medium. The 1 ml of cells was concentrated and plated on -uracil, -leucine, +raffinose, +galactose to induce cutting by CRISPR/Cas9 and to select for uracil prototrophy. After restreaking colonies on–uracil plates, clones were expanded in liquid –uracil medium for freezer stocks and CHEF-gel plugs.

Agarose plugs for CHEF gel electrophoresis were generated using the method by L. Argueso (described in [ 23 ]) or by an adaptation of the method by S. Iadonato and A. Gnirke (described in [ 24 ]). Run conditions in the BioRad CHEF-DRII were 0.8% LE agarose in 0.5XTBE in 2.3 L 0.5% TBE running buffer at 14°C. Switch times were 47” to 170” at 165V for 39–62 hours. Standard conditions for Southern blotting and hybridization with 32 P-labeled PCR probes are described by Tsuchiyama et al. [ 24 ]. Hybridization intensity was determined using a BioRad Personal Molecular Imager. Primer pairs used to create 32 P-labeled PCR probes are given in S2 Table .

Small independent colonies of the split-ura3 strain were picked from a synthetic complete plate with 5-FOA and inoculated in 1 ml of complete synthetic yeast medium and grown to a saturation density of ~10 8 cells. The 1 ml of cells was concentrated by centrifugation, plated onto a single -uracil plate, and incubated at 30°C for 3–5 days. A maximum of two colonies from each -uracil plate were restreaked on a fresh -uracil plate. Each purified clone was grown in 8 ml of -uracil liquid medium to make freezer stocks, CHEF-gel plugs and “NIB-n-grab” DNA preps ( https://fangman-brewer.genetics.washington.edu/nib-n-grab.html ; see below). To test for the effect of nucleotide depletion on the formation of Ura+ clones, the 1 ml of medium contained either 50 or 200 mM HU.

In the following diagrams black lines are used to indicate the original chromosomal sequences and blue lines refer to the ODIRA generated intermediates and their fate after recombination with chromosomes. (A) Sites on chromosomes II and IX were modified by insertion of overlapping fragments of the URA3 gene. A probe to the unique 5’ portion of the SUL1 fragment used for Southern blotting is highlighted in cyan. (B) Recombination between the two marked chromosomes can recreate an intact URA3 gene but results in the creation of an unstable dicentric Ura+ chromosome. Deletion of one of the centromeres results in stabilized chromosome. The reciprocal product is an acentric chromosome that is lost during mitosis. (C) The chromosome II ura fragment, amplified as an extrachromosomal, inverted circular molecule, can recombine with the target site (ra3) on chromosome IX to generate a functional URA3 gene. The resulting copy of chromosome IX contains a tandem inverted triplication of the ra segment. (D) A single replication error at the centromere-proximal fork generates a palindromic linear fragment. Recombination between one of the copies of the ura fragments and the target ra3 fragment on chromosome IX re-forms a functional URA3 gene and creates a translocation between the palindromic chromosome and chromosome IX. To cover the loss of essential genes on the left arm of chromosome IX, an unrearranged chromosome IX is also expected. (E) Anticipated CHEF gel results for the four strains described in A-D. The ethidium bromide stained gel reveals all chromosomes—the rearranged chromosome for each case is indicated in red. Hybridization with chromosome specific probes is used to distinguish the different outcomes, including the 5’SUL1 probe shown in (A). The relative intensities of the 5’SUL1 probe are indicated for each band as either 1 or 2 (green type). The deleted CEN2 (B) is often retained as a large circular molecule through recombination between flanking TY repeated elements. In CHEF gels, these circular molecules are often found retained in the well. Dotted rectangles in B, C, D show the expected final products.

BY4741 (MATa, his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and BY4742 (MATα, his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) were used to construct the split-ura3 strain. The two partially overlapping regions of the URA3 gene, referred to as ura and ra3, were generated in two steps. We first selected (on -uracil plates) for URA3 insertion on chromosome II and chromosome IX, respectively into BY4741 and BY4742, by transformation with a PCR fragment derived from pRS406 with 100 bp homology arms ( S1 Table ; SUL1_URA3_F and SUL1_URA3_R; Chr9_URA3_Chr9_F and Chr9_URA3_Chr9_R). These strains were then transformed with truncated PCR fragments of ura and ra3, similarly created from pRS406 and primers with the same homology arms ( S1 Table ; SUL_URA3_F and ura_SUL1_R; Chr9_ra3_F and Chr9_URA3_Chr9_R). Transformants were selected on plates with 5-fluoro orotic acid (5-FOA) and confirmed by PCR/Sanger sequencing and Southern blotting of CHEF gels. BY4741 containing the ura fragment was mated to BY4742 containing the ra3 fragment to create the doubly heterozygous diploid. Sporulation and tetrad dissection resulted in a haploid spore, s2-1 (MATa, ura3Δ, his3Δ1, leu2Δ0, lys2Δ0, sul1::ura, FAT1-3’::ra3; hereafter referred to as the “split-ura3 strain”; Fig 2A ), with both partial ura3 fragments that were confirmed by CHEF gel/Southern blotting. This strain was used in all subsequent experiments involving selection for Ura+ clones. The ura insert lies within the SUL1 gene between coordinates 789418 and 791405 on chromosome II on the Watson strand. The ra3 fragment lies in an intergenic region between FAT1 and CST26 at coordinates 321188–321194 on chromosome IX, also on the Watson strand ~34 kb upstream of CEN9 (355629–355745). The overlap between ura and ra3 is 203 bp.

Results

Reducing dNTP levels through inhibition of ribonucleotide reductase reduces the relative frequency of inverted Ura+ clones Growing yeast cells in hydroxyurea reduces nucleotide pools [30] and alters features of the replication fork, including a ~16-fold reduction in fork speed [31], uncoupling of the replicative helicase (CMG) from the replisome [32], and an increase in the length of the single stranded gap [33]. The increased persistence and length of the single stranded regions increase the probability of single stranded breaks at forks, which would result in a single-ended double stranded break (Fig 4A). Because the broken dsDNA has no partner it cannot be repaired by end-joining mechanisms. However, the single-ended breaks are competent for BIR or homologous recombination and may be responsible for the increase in S-phase-specific homologous recombination events seen in HU treated cells [34]. In the split-ura strain such breaks at the telomere adjacent fork produce an end that can invade the homology on chromosome IX and generate the Ura+ clones through direct recombination or BIR. Single stranded breaks at the centromeric adjacent fork cannot produce Ura+ clones through these mechanisms. Nevertheless, end resection and fold over of breaks at the centromere-adjacent fork could produce a hairpin intermediate—the same intermediate we propose is produced by ODIRA (Fig 4A) that gives rise to Ura+ recombinants. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Induction of DNA double stranded breaks by CRISPR/Cas9 cleavage centromere-proximal to 5’ura (SUL1) eliminates inverted amplicons. (A) The proposed hairpin intermediate could be formed (left to right) by a break in the single stranded DNA at a fork, by a double-stranded DNA break in nonreplicating DNA or by a single Cen-proximal ODIRA template-switching event. (B) CRISPR/Cas9 was used to induce a DSB either centromere-proximal (708 kb) or telomere-proximal (792 kb) to the ura locus. Cells that had been transformed with the CRISPR/Cas9 plasmid (S1 Fig) were selected for on –leucine plates. Twenty independent transformants were grown to saturation in 1 ml of liquid –leucine medium and the entire saturated cultures (approximately 108 cells) were spread on–uracil plates (-leucine, +galactose, +rafinose) to induce Cas9 expression and to select for successful regeneration of a functional URA3 gene. (C) Cutting at 792 kb greatly increased Ura+ colony frequency while cutting at 708 resulted in no Ura+ colony recovery. (D) Cutting at 792 resulted in direct recombination events between chromosome II and IX, while cutting at 708 appeared to interfere with the ability to recreate the functional URA3 gene. The control plasmid lacking a guide RNA gene produced a similar ratio of hairpin URA3 chromosomes to recombined chromosomes (6:14) as found for the non-transformed split-ura3 haploid strain (11:39). https://doi.org/10.1371/journal.pgen.1010850.g004 Growing the split-ura strain in the presence of hydroxyurea allows us to distinguish between ODIRA and single-ended double stranded break generation of Ura+ clones (Fig 2A). In the HU grown cultures, we recovered a roughly 3.5 to 5-fold higher frequency of Ura+ clones; however, examination of the clones revealed that 73 of the 76 clones were due to direct recombination—the type produced by a telomere-proximal fork break. Only 3 Ura+ events (2 of 43 clones grown in 50 mM HU and 1 of 33 clones grown in 200 mM HU; clone HU++3 in S3 Fig was the single clone recovered from growth in 200 mM HU) were events that could be attributed to hairpin intermediates. If single-ended DNA breaks were responsible for the formation of inverted neochromosomes, we would have expected to see a similar 3.5 to 5-fold increase in the inverted events. These results suggest that inverted neochromosomes are not created by repair of a single-ended DNA intermediate.

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