Patching Broken Chromosomes with Extranuclear Cellular DNA Xin Yu, Abram Gabriel  Molecular Cell  Volume 4, Issue 5, Pages 873-881 (November 1999) DOI: 10.1016/S1097-2765(00)80397-4

Figure 1 The Experimental System (A) Structure of the URA3 allele on S. cerevisiae chromosome V, generated for these experiments. Note that the presence or absence of the HO cut site, derived from the MATa locus, distinguishes single cut site from no cut site strains. The position of various oligonucleotide primers (numbers and half arrows) used for PCR and sequencing are shown. (B) The actin intron placed into URA3 is normally spliced, resulting in uracil prototrophy (Ura+) and sensitivity to the drug 5-fluoro orotic acid (FOAS). After creating a DSB within the engineered actin intron with HO endonuclease, cells either die, are repaired in a way allowing normal splicing, or are repaired in a way that prevents splicing. The latter situation leads to a phenotype of uracil auxotrophy (Ura−) and resistance to 5-FOA (FOAR). (C) Various plasmid constructs consisting of a centromere-based HIS3-containing plasmid vector (pRS413), the same vector containing the 309 base URA3::actin intron cassette (AGE1638), the same vector containing the 438 base URA3::actin intron::HO cut site cassette (AGE1662), or four additional constructs with increasingly larger inserts within the URA3::actin intron cassette were transformed into strain YH50, which is auxotrophic for both histidine and uracil, and then grown on synthetic complete media lacking histidine (SC-his), synthetic complete media lacking histidine and uracil (SC-his-ura), and synthetic complete media lacking histidine but supplemented with 1 mg/ml 5-FOA (FOA-his). Molecular Cell 1999 4, 873-881DOI: (10.1016/S1097-2765(00)80397-4)

Figure 2 Analysis of Ty1 Insertions into a Chromosomal Break (A) Schematic drawing showing the position and length of each Ty1 cDNA insertion, relative to a complete Ty1 element. LTRs refer to the near-identical long terminal repeats present at the 5′ and 3′ ends of Ty1. Each LTR consists of three regions, separated by vertical lines: U3 (1–240 or 5585–5824), R (241–288 or 5825–5872), and U5 (289–334 or 5873–5918). The ten base primer binding site (PBS) region (335–344) borders the 5′ U5 and is complementary to the 3′ end of the yeast initiator methionine tRNA. Squiggly lines downstream of the 3′ LTR refer to extra bases corresponding to the PBS that were found in nearly all inserts. Double slashes within insertions in the 5′ LTR refer to discontinuities found in three compound insertions, which most likely represent aberrant strand jumps from the minus strand strong stop intermediate to internal locations within Ty1 (arrow) rather than to the expected 3′ R region. The reference number for each insertion associated with the 5′ LTR is shown, corresponding to the detailed structures in (B). Insertions associated with the 3′ LTR are drawn according to increasing size, as they are listed in (B). (B) The structure of each Ty1 cDNA insertion identified at the DSB site. The sequence of the HO cut site is shown, with the resulting 3′ overhanging terminal TGTT shown on both sides of the cut. The U5/PBS border sequence is shown for comparison with insert junctions. Uppercase bases are preexisting MATa sequences around the HO cut site. Lowercase bases are inserted Ty1 sequences. Underlined bases are overlaps between two noncontiguous DNA segments. Bold bases are presumed nontemplated junctional sequences (ntb). Italicized bases are mismatched bases within an overlap region. Gaps are included for the purpose of alignment. Numbering of each insertion refers to the sequence of the reference Ty1 element Ty1-H3 (Genbank accession number M18706). Length of insertion includes overlapping bases. Simple (i.e., continuous sequence) insertions are divided into two groups based on whether the 3′ end or 5′ end of Ty1 is involved in the insertion. Within the first group, three insertions (y625, y789, and y1483) are oriented opposite to the others. Compound insertions comprise two discontinuous sequences joined by overlapping sequences. Molecular Cell 1999 4, 873-881DOI: (10.1016/S1097-2765(00)80397-4)

Figure 3 Analysis of Mitochondrial DNA Insertions into a Chromosomal Break (A) Each flag marks the position, along the 85,779 bp circular mitochondrial genome, of a DNA segment identified at the DSB. Black versus white flags refer to the orientation of the mitochondrial DNA relative to the HO cut site. The location of each flag around the circle corresponds to the nucleotide position listed in (B). (B) The structure of each mitochondrial insertion identified at the DSB site. The same conventions used in Figure 2B apply to these insertions. Lowercase bases are inserted mitochondrial DNA sequences. For compound elements, a slash represents a nonoverlapping border between two noncontiguous DNA segments. Numbering of insertions and location relative to mitochondrial landmarks is based on the complete S. cerevisiae mitochondrial genome sequence reported by Foury et al. 1998. (C) Examples of the complete sequence of the five shortest mitochondrial DNA insertions. Note that Gs and Cs make up only 5% of the total inserted bases. Molecular Cell 1999 4, 873-881DOI: (10.1016/S1097-2765(00)80397-4)