Long-term growth in selective medium selects for integration of plasmid sequences.

<p>After 80 to 90 generations of selective growth in–uracil medium, single clones were isolated from the cultures of transformants 1, 3, 5, 7, and 8. DNA from the first day (F), the last day (L) and the clone (C) was isolated by smash-and-grab and by an agarose plug method and analyzed by conventional (A) and CHEF (B) gel electrophoresis. The fates of the transforming <i>URA3-ARS1</i> dog bones were determined by Southern blots using <i>URA3</i> as a probe (ethidium bromide images on left, Southern hybridization on right). The parental strain BY4741 (P) has a complete deletion of <i>URA3</i> on chromosome V. (A) Plasmids were identified in all samples (nicked circular (NC) and supercoiled (SC) molecules of two different sized plasmids), with the exception of the clone from transformant #7 (red box). In this sample, hybridization to chromosomal fragments suggested that the plasmid had integrated (marked with *). (B) CHEF analysis confirms the presence of <i>URA3</i> on chromosome IV. Increased hybridization of <i>URA3</i> sequences (over the background signal found in the parental strain (P)) on the last days of selective growth suggests that integration had occurred in other cultures, although the single clones did not reflect this event. (Note that small plasmids migrate very slowly under CHEF gel electrophoresis conditions and also are trapped in the wells. These molecules are indicated by brackets on the right side of the gel.) Both DNA sampling techniques also revealed that free plasmids were experiencing other changes as a consequence of long-term selective growth.</p

Ligation of leading and lagging “nascent” strands at a replication fork <i>in vitro</i>.

<p>(A) Each oligonucleotide was separately analyzed by electrophoresis in a 3.75% agarose gel after boiling and chilling on ice to allow the hairpins on both oligos to fold (leading strand marked with red arrow, lagging strand marked with blue arrow). The differences in migration of these nearly identically sized oligonucleotides are likely due to their different three-dimensional structures. The two oligonucleotides were mixed (in nearly equal molar amounts), boiled, chilled on ice and then returned to room temperature to allow the two complementary, single stranded tails to anneal. The image of the ethidium bromide stained gel reveals that some unreacted lagging strands remain (blue bar), but the majority of the oligonucleotides have formed higher molecular weight molecules (extremes highlighted with purple bars; intermediates from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.g002" target="_blank">Fig 2</a> are labeled B, C, and D). The differences in migration of these molecules with identical mass are likely due to different branching patterns. Addition of DNA Ligase + ATP (last lane on the gel) results in a slightly faster migration of the slowest migrating form (orange bar) relative to its counterpart in the no-ligase lane. (B) Reserved samples from the last two lanes in (A) were used as templates in PCR reactions to detect a covalent linkage between the two oligonucleotides across the fork. PCR primers were designed such that their 3’ends resided in the single stranded linkers at the distal arms of the leading and lagging strands (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.g002" target="_blank">Fig 2C</a>). The image of the ethidium bromide stained 3.75% agarose gel reveals a PCR product of 78 bp that can be cleaved by <i>Xho</i>I into 47 and 31 bp fragments (gray arrows).</p

Transformation of yeast with an artificial dog bone results in inverted dimeric plasmids.

<p>(A) A <i>Hind</i>III-<i>Xba</i>I restriction fragment from plasmid pUA-DirB contains the <i>URA3</i> gene from chromosome V (blue arrow) and the origin <i>ARS1</i> from chromosome IV (red circle). Oligonucleotide hairpins were designed with <i>Hind</i>III (blue) and <i>Xba</i>I (brown) overhangs, 5’-phosphates to ensure their ligation to the ends of the <i>URA3-ARS1</i> fragment, and additional restriction sites in the duplex and loop portions to facilitate analysis of the DNA after transformation. (B) Map of the anticipated inverted dimeric plasmid after transformation and replication in yeast. Cleavage sites are indicated in bp. (C) Eight transformants were chosen at random; all eight had plasmids and five (transformants 1, 3, 5, 7, and 8) had restriction maps that were consistent with the map expected for a circular inverted dimer. The Southern blot using a <i>URA3</i> probe for transformant #3 is shown.</p

The inverted dimeric plasmid integration preserves both palindromic junctions, creating an inverted triplication of <i>ARS1</i>.

<p>(A) The map illustrates the expected structure after homologous integration of the dimeric plasmid between <i>TRP1</i> and <i>GAL3</i> on chromosome IV. The positions and sizes of <i>Bgl</i>II fragments, as well as probe sequences are illustrated. (B) A snap-back assay detects palindromic sequences by virtue of their ability to recreate duplex fragments of half the original size after boiling and quick cooling on ice. Additionally, these sequences are resistant to the ssDNA nuclease S1. (C and D) Southern blots of snap-back assays on the two palindromic junctions of the integrated plasmid in the last day clone from transformant #7. Lane 1 = native duplex <i>Bgl</i>II fragments; lane 2 = denatured/renatured palindromic duplexes; lane 3 = S1 nuclease resistant duplex DNA. The probe in (C) detects the <i>URA3</i> palindromic fragment; the probe in (D) detects the <i>ARS1</i> palindromic fragment as well as the non-inverted copy of <i>ARS1</i> adjacent to <i>GAL3</i>. Note that the larger ssDNA fragment (in lane 2) is degraded by S1 (lane 3).</p

An <i>in vitro</i> construct to test for fork reversal and cross-strand ligation.

<p>An artificial replication fork was generated using commercially synthesized oligonucleotides (sizes of 100 and 99 nucleotides, with a 5’-phosphate on the 99 nucleotide lagging oligonucleotide). (A) To facilitate assembly of the fork from its component strands, the “parental” and “nascent” strands of the leading arm of the duplex, and likewise, the two strands of the lagging arm of the replication fork were covalently joined by a short linker. Complementary sequences in the two, 30 base, single stranded tails allow the fork to assemble <i>in vitro</i>. Features engineered into the two oligos include a 7 bp inverted repeat (blue sequences highlighted with small arrows) separated by 11 nucleotides of non-complementary sequence, and various restriction sites to facilitate evaluation of the fork and its products. (B) The single stranded stretch on the lagging strand exposes two complementary copies of the inverted repeat (in blue with arrow) which are expected to fold into a stem-loop structure to achieve the lowest free energy state for the forked molecule. (C and D) Breathing of the duplexes at the fork facilitates fork reversal and generates alternative conformations by branch migration—all with identical numbers of paired bases. The structures in B and D reflect the two extreme branch-migrated forms, with C representing only one of 25 possible intermediates. The two nucleotides in red are brought into proximity in the branch-migrated forms, providing a substrate for ligation by DNA ligase. Completion of the ligation step creates an <i>Xho</i>I restriction site. PCR primers (shown in C) target the two linkers.</p

<i>SUL1</i> amplicon structures generated in the presence and absence of <i>ARS228</i>.

<p>(A) Top: Seven sulfate limited chemostats were grown for ~200 generations with a strain deleted for <i>ARS228</i>. A single random clone was isolated from each chemostat and characterized by <i>ApaL</i>I snap-back assays (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.s007" target="_blank">S5 Fig</a>) and aCGH (the amplification junctions are marked by brackets above the dotted line, aligned with the chromosomal map of the right arm of chromosome II from coordinates 750 kb to telomere (813 kb)). Among the single clones (S10201 to 7) we identified four clones with interstitial inverted amplification events (blue brackets) and three clones which were inconsistent with inverted amplification structures (black brackets; 10202-c1474 also suffered a telomere deletion). Additional unique clones (designated as cA-cE, below the dotted line) were analyzed from each chemostat culture. Among the seven additional clones, one clone was a complex isochromosome with deletion of one copy of <i>CEN2</i> (green clone). The junction sequence (green square) was not pursued further. (A) Bottom: Wild type cultures (<i>ARS228</i>) selected from the sulfate-limited chemostat cultures (~200 generations) were pooled from published work from our labs and their amplification junctions are shown below the chromosome map (red brackets). Molecular characterizations of chromosome II structures in these clones are consistent with interstitial inverted amplification. Pop 2–7 clones are from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.ref004" target="_blank">4</a>]; B9, 10, 11, and 13 are from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.ref018" target="_blank">18</a>]; and s1c2 and s2c1 are from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.ref016" target="_blank">16</a>]. The vertical orange bars mark the positions of known ARSs. (B) Sizes of amplified regions from the <i>ARS228</i> and <i>ars228Δ</i> strains generate medians of 16.5 and 28.9 kb, respectively, with a Wilcoxon Mann Whitney significance p-value of 0.027.</p

PCR amplification across the <i>URA3-GAL3</i> junction confirms plasmid integration adjacent to <i>ARS1</i> on chromosome IV.

<p>(A) The map illustrates the region of homology (shaded in grey) shared between chromosome IV (top; genomic region from <i>SOK1</i> to <i>SNQ2</i>—coordinates 460 to 467 kb—taken as a screen shot from the <i>Saccharomyces</i> genome database; <a href="http://www.yeastgenome.org/" target="_blank">www.yeastgenome.org</a>) and the plasmid sequences (bottom). The position of PCR primers (red arrows) predicts a 1.2 kb fragment for integrants that have recombined by homology in the <i>TRP1</i>-<i>GAL3</i> region. (B) Ethidium bromide stained gel of PCR fragments. The presence of 1.2 kb PCR fragments in each of the last day samples (L) and clone (C) from the last day of transformant #7 confirms the integration site of the plasmid sequences.</p

Key steps in the origin-dependent inverted-repeat amplification model for the generation of interstitial, inverted triplications.

<p>(1) Replication forks initiated at an origin of replication (vertical blue line) undergo fork reversal at small, interrupted inverted repeats (small blue/purple arrows; a and a’) leading to a replication error that covalently links nascent leading and lagging strands. These self-complementary DNA loops are expelled by adjacent replication forks to generate free linear DNA duplexes with closed loops (“dog bone” molecules) that correspond to the interrupted, inverted repeats and their intervening sequences. (2) In the next cell cycle, replication from the origin on the linear fragment generates a circular plasmid with the two copies of the amplified region in inverted orientation with the interrupted, inverted repeats at the head-to-head and tail-to-tail junctions. (3) Integration of the plasmid into the chromosomal locus generates the triplication with the central repeat in reverse orientation. The drawings are adapted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.g002" target="_blank">Fig 2</a> in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005699#pgen.1005699.ref002" target="_blank">2</a>].</p