Chromosome Fusions following Telomere Loss Are Mediated by Single-Strand Annealing  Xiaorong Wang, Peter Baumann  Molecular Cell  Volume 31, Issue 4, Pages 463-473 (August 2008) DOI: 10.1016/j.molcel.2008.05.028 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Genetic Requirements for Chromosome Circularization and Survival with Circular Chromosomes (A) NotI-digested chromosomes were analyzed by pulsed-field gel electrophoresis and Southern blotting. The terminal fragments of chromosome I and II are detected using a mixture of four probes. (B) Survival of double mutants in presence or absence of pot1+ plasmid (pJB20 or pXW14). Cells of the indicated genotypes were plated on selective and counterselective media to assay growth in the presence and absence of plasmid-borne pot1+. (C) Spotting assay of 10-fold serial dilutions of cells. The plasmid is retained on EMM HUA and selected against on YES plus 5FUdR. (D) Synthetic lethality of pot1− rad11-D223Y mutation following loss of pot1+ plasmid. Cells of the indicated genotypes were plated to YES (5FUdR) for counterselection. Molecular Cell 2008 31, 463-473DOI: (10.1016/j.molcel.2008.05.028) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Mapping and PCR Amplification of Chromosome Fusions (A) Schematic of terminal region on chromosomes I and II based on assembly of pNSU70, pNSU56, pB15E1, and part of cos212 (AL391746). The assembled sequence is shown in Figure S3. Individual sequences for pNSU56, pNSU70, and pB15E1 are available from the Wellcome Trust Sanger Institute. Arrows and numbers indicate orientation and position of primers, with each primer name reflecting the distance from the telomeric end. The positions of the Tlh helicase and telomere-associated sequences (TAS) are indicated with black bars. Two groups of homology boxes (H1–H5, H1′–H5′) are present on each chromosome arm in inverted orientation as indicated by arrows. (B) Identification of sequences absent in strains with circular chromosomes. Locations of regions amplified by PCR are shown as gray bars in (A). Different lanes represent independently derived pot1− strains based on PP164 (lanes 1–6) and BG_1225 (lanes 9–13). Primer sequences are available as Supplemental Data. (C) Schematic of chromosomes and orientation of PCR primers for amplification of junctions in circular chromosomes. (D) Amplification of DNA fragments flanking the junctions of circular chromosomes. Primers 7926 and 31268 were used with genomic DNA from pot1− cells in PCR reactions. Different lanes represent independently derived pot1− strains based on BG_1225 (A to F) and PP164 (G to I). Molecular Cell 2008 31, 463-473DOI: (10.1016/j.molcel.2008.05.028) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Junctions of Circular Chromosomes (A) Schematic of two chromosome ends in head-to-head orientation. Fusions occur between a homology box in a telomere proximal set (H1–H5) and the homologous region in the telomere distal set (H1′–H5′) of the other chromosome arm. Arrows are used to illustrate the matching orientation of repeats for single-strand annealing. (B) Schematic of chromosome fusions involving homology regions H1 and H1′ (upper) or H3 and H3′ (lower). Sequences that are lost prior to fusion are in gray. The positions and orientations of PCR primers 7926 and 31268 used to amplify the junction are shown. Alignments are available as Supplemental Data. (C) Sequences surrounding junctions of fused chromosome ends that use homology regions four and five were amplified with primers 14206 and 31268 (upper panel) and 14206 and 28900 (lower panel). (D) Schematic of chromosome fusions involving homology regions H4/H4′ or H5/H5′. Molecular Cell 2008 31, 463-473DOI: (10.1016/j.molcel.2008.05.028) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Southern Blot of EcoRI-Digested Genomic DNA Samples Top panel probed with an oligonucleotide probe that hybridizes between H2 and H3. Middle panel probed for telomeric repeats and lower panel probed for rad16+ as loading control (LC). Letters refer to independent isolates of pot1− strains as in Figure 2. Molecular Cell 2008 31, 463-473DOI: (10.1016/j.molcel.2008.05.028) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Deletion of rad16+ Compromises Survival in the Absence of Trt1 (A) Spores derived by tetrad dissection from a trt1+/− rad16+/− strain give rise to colonies of all four genotypes. (B) Cells of the indicated genotypes were subjected to serial restreaks. Survivor formation is compromised in trt1− rad16− strains. (C) Telomeric Southern blot showing progressive telomere shortening in trt1− and trt1− rad16− cells during growth in liquid culture. Cultures were diluted daily to a density of 2.5 × 104 cells/ml. (D) Chromosome fusions and circularization in trt1− and trt1− rad16− analyzed by pulsed-field gel electrophoresis as in Figure 1. Inter- and intrachromosomal fusions are marked by arrows. Molecular Cell 2008 31, 463-473DOI: (10.1016/j.molcel.2008.05.028) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Rad16 Is Not Required for NHEJ-Mediated Chromosome Fusions PFG analysis of cells incubated either in YES (Complete) or in EMM (− Nitrogen) for 48 hr. Bands corresponding to chromosome fusions are indicated by arrows. Molecular Cell 2008 31, 463-473DOI: (10.1016/j.molcel.2008.05.028) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Effect of Taz1 and Initial Telomere Length on Chromosome Fusions following Loss of Pot1 (A) Telomeric Southern blot of genomic DNA digested with EcoRI. (Lanes 1 to 4) Haploid progeny of a pot1+/− taz1+/− strain (D1). (Lanes 5 to 13) Haploid progeny of a pot1+/− taz1−/− strain (D2). Lanes represent individual isolates from random spore analysis. Telomeric hybridization in defined bands is observed for pot1− taz1− strains derived from D2, but not from D1. (B) Genomic DNA of the same strains as in (A) was digested with NotI and analyzed by PFGE and Southern blotting with proximal probes against the C, I, L, and M fragments. Products of chromosome circularization (C+M and I+L) are indicated on the right. (C) Same blot as in (B) but hybridized with a telomeric probe. Telomeric DNA is retained in all I+L and several C+M fusion bands. Molecular Cell 2008 31, 463-473DOI: (10.1016/j.molcel.2008.05.028) Copyright © 2008 Elsevier Inc. Terms and Conditions