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DNA bridging of yeast chromosomes VIII leads to near-reciprocal translocation and loss of heterozygosity with minor cellular defects

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Abstract

Loss of heterozygosity (LOH) of tumor suppressor genes in somatic cells is a major process leading to several types of cancer; however, its underlying molecular mechanism is still poorly understood. In the present work, we demonstrate that a linear DNA molecule bridging two homologous chromosomes in diploid yeast cells via homologous recombination produce LOH-generating regions of hemizygosity by deletion. The result is a near-reciprocal translocation mutant that is characterized by slight cell cycle defects and increased expression of the multidrug-resistant gene VMR1. When the distance between target regions is approximately 40 kb, the specificity of gene targeting becomes less stringent and an ensemble of gross chromosomal rearrangements arises. These heterogeneous genomic events, together with the low frequency of specific translocation, confirm that several pathways contribute to the healing of a broken chromosome and suggest that uncontrolled recombination between parental homologs is actively avoided by the cell. Moreover, this work demonstrates that the common laboratory practice of making targeted gene deletions may result in a low, but not negligible, frequency of LOH due to the recombination events triggered between homologous chromosomes in mitosis.

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References

  • Bruschi CV, Howe GA (1988) High frequency FLP-independent homologous DNA recombination of 2μ plasmid in the yeast Saccharomyces cerevisiae. Curr Genet 14:191–199

    Article  PubMed  CAS  Google Scholar 

  • Cahill D, Connor B, Carney JP (2006) Mechanisms of eukaryotic DNA double strand break repair. Front Biosci 11:1958–1976

    Article  PubMed  CAS  Google Scholar 

  • Chenevert JM, Naumovski L, Dumas LB, Friedberg EC (1984) Use of plasmid-mediated resistance to the antibiotic G418 for the rapid screening of a yeast mutant library. J Exp Pathol 1:307–313

    PubMed  CAS  Google Scholar 

  • Chizhikov V, Chikina S, Gasparian A, Zborovskaya I, Steshina E, Ungiadze G, Samsonova M, Chernyaev A, Chuchalin A, Tatosyan A (2002) Molecular follow-up of preneoplastic lesions in bronchial epithelium of former Chernobyl clean-up workers. Oncogene 21:2398–2405

    Article  PubMed  CAS  Google Scholar 

  • Daigaku Y, Mashiko S, Mishiba K, Yamamura S, Ui A, Enomoto T, Yamamoto K (2006) Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle. Mutat Res 600:177–183

    PubMed  CAS  Google Scholar 

  • Gjuracic K, Pivetta E, Bruschi CV (2004) Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots. Mol Genet Genom 271:437–446

    Article  CAS  Google Scholar 

  • Gray M, Honigberg SM (2001) Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces. Nucleic Acids Res 29:5156–5162

    Article  PubMed  CAS  Google Scholar 

  • Haigis KM, Dove WF (2003) A Robertsonian translocation suppresses a somatic recombination pathway to loss of heterozygosity. Nat Genet 33:33–39

    Article  PubMed  CAS  Google Scholar 

  • Haigis KM, Caya JG, Reichelderfer M, Dove WF (2002) Intestinal adenomas can develop with a stable karyotype and stable microsatellites. Proc Natl Acad Sci U S A 99:8927–8931

    PubMed  CAS  Google Scholar 

  • Hastings PJ, McGill C, Shafer B, Strathern JN (1993) Ends-in vs. ends-out recombination in yeast. Genetics 135:973–980

    PubMed  CAS  Google Scholar 

  • Inbar O, Kupiec M (1999) Homology search and choice of homologous partner during mitotic recombination. Mol Cell Biol 19:4134–4142

    PubMed  CAS  Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    PubMed  CAS  Google Scholar 

  • Janoueix-Lerosey I, Hupe P, Maciorowski Z, La Rosa P, Schleiermacher G, Pierron G, Liva S, Barillot E, Delattre O (2005) Preferential occurrence of chromosome breakpoints within early replicating regions in neuroblastoma. Cell Cycle 4:1842–1846

    PubMed  CAS  Google Scholar 

  • Kaiser C, Michaelis S, Mitchell A (1994) Preparation of chromosome-sized yeast DNA molecules in solid agarose. In: CSH Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 179–180

    Google Scholar 

  • Kool M, de Haas M, Scheffer GL, Scheper RJ, van Eijk MJ, Juijn JA, Baas F, Borst P (1997) Analysis of expression of cMOAT (MRP2), MRP3, MRP4, andMRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res 57:3537–3547

    PubMed  CAS  Google Scholar 

  • Lander JK, Fan H (1997) Low-frequency loss of heterozygosity in Moloney murine leukemia virus-induced tumors in BRAKF1/J mice. J Virol 71:3940–3952

    PubMed  CAS  Google Scholar 

  • Langston LD, Symington LS (2004) Gene targeting in yeast is initiated by two independent strand invasions. Proc Natl Acad Sci U S A 101:15392–15397

    Article  PubMed  CAS  Google Scholar 

  • Moore JK, Haber JE (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 16:2164–2173

    PubMed  CAS  Google Scholar 

  • Morrow DM, Connelly C, Hieter P (1997) “Break copy” duplication: a model for chromosome fragment formation in Saccharomyces cerevisiae. Genetics 147:371–382

    PubMed  CAS  Google Scholar 

  • Nicolini C (2006) Nanogenomics for medicine. Nanomed 1:147–52 Review

    Article  PubMed  Google Scholar 

  • Nikitin D, Tosato V, Zavec AB, Bruschi CV (2008) Cellular and molecular effects of non-reciprocal chromosome translocations in S. cerevisiae. Proc Natl Acad Sci 105:9703–9708

    Article  PubMed  CAS  Google Scholar 

  • Norris MD, Smith J, Tanabe K, Tobin P, Flemming C, Scheffer GL, Wielinga P, Cohn SL, London WB, Marshall GM, Allen JD, Haber M (2005) Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro. Mol Cancer Ther 4:547–553

    Article  PubMed  CAS  Google Scholar 

  • Pan H, Califano J, Ponte JF, Russo AL, Cheng KH, Thiagalingam A, Nemani P, Sidransky D, Thiagalingam S (2005) Loss of heterozygosity patterns provide fingerprints for genetic heterogeneity in multistep cancer progression of tobacco smoke-induced non-small cell lung cancer. Cancer Res 65:1664–1669

    Article  PubMed  CAS  Google Scholar 

  • Reifenberger J, Reifenberger G, Liu L, James CD, Wechsler W, Collins VP (1994) Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p. Am J Pathol. 145:1175–1190

    PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Analysis of genomic DNA by Southern hybridization. In: CSH Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 9.31–9.58

    Google Scholar 

  • Smith CE, Llorente B, Symington LS (2007) Template switching during break-induced replication. Nature 447:102–105

    Article  PubMed  CAS  Google Scholar 

  • Stark JM, Jasin M (2003) Extensive loss of heterozygosity is suppressed during homologous repair of chromosomal breaks. Mol Cell Biol 23:733–743

    Article  PubMed  CAS  Google Scholar 

  • Storici F, Coglievina M, Bruschi CV (1999) A 2-micron DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae. Yeast 15:271–283

    Article  PubMed  CAS  Google Scholar 

  • Tosato V, Waghmare SK, Bruschi CV (2005) Non-reciprocal chromosomal bridge-induced translocation (BIT) by targeted DNA integration in yeast. Chromosoma 114:15–27

    Article  PubMed  CAS  Google Scholar 

  • Umezu K, Hiraoka M, Mori M, Maki H (2002) Structural analysis of aberrant chromosomes that occur spontaneously in diploid Saccharomyces cerevisiae: retrotransposon Ty1 plays a crucial role in chromosomal rearrangements. Genetics 160:97–110

    PubMed  CAS  Google Scholar 

  • Wach A, Brachat A, Pohlmann R, Philippsen P (1999) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 410:1793–1808

    Google Scholar 

  • Waghmare SK, Bruschi CV (2005) Differential chromosome control of ploidy in the yeast Saccharomyces cerevisiae. Yeast 22:625–639

    Article  PubMed  CAS  Google Scholar 

  • Waghmare SK, Caputo V, Radovic S, Bruschi CV (2003) Specific targeted integration of kanamycin resistance-associated non-selectable DNA in the genome of the yeast Saccharomyces cerevisiae. Biotechniques 34:1024–1028

    PubMed  CAS  Google Scholar 

  • Wiese C, Gauny SS, Liu WC, Cherbonnel-Lasserre CL, Kronenberg A (2001) Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor. Cancer Res 61:1129–1137

    PubMed  CAS  Google Scholar 

  • Wijnhoven SW, Sonneveld E, Kool HJ, van Teijlingen CM, Vrieling H (2003) Chemical carcinogens induce varying patterns of LOH in mouse T-lymphocytes. Carcinogenesis 24:139–144

    Article  PubMed  CAS  Google Scholar 

  • Zang Y, Garrè M, Gjuracic K, Bruschi CV (2002) Chromosome V loss due to centromere knockout or MAD2 deletion is immediately followed by restitution of homozygous diploidy in Saccharomyces cerevisiae. Yeast 19:553–564

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Sanjeev Waghmare for his help in labeling the haploid strains with fluorescent markers, Erica Faverio Margoni for the analyses with the confocal microscopy and Mike Jewitt for the assistance in the screening of the ΔDUR3 transformants. This project was also supported by grants to Fondazione Elba by MIUR for “Funzionamento” and by a FIRB International Grant on Proteomics and Cell Cycle (RBIN04RXHS) from MIUR (Ministero dell’Istruzione, Università e Ricerca) to CIRSDNNOB-Nanoworld Institute of the University of Genova.

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Authors and Affiliations

  1. ICGEB Yeast Molecular Genetics Laboratory, AREA Science Park, Padriciano 99, I-34012, Trieste, Italy

    Valentina Tosato & Carlo V. Bruschi

  2. Nanoworld Institute, University of Genova, Corso Europa 30, I-16132, Genova, Italy

    Claudio Nicolini

Authors
  1. Valentina Tosato
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  2. Claudio Nicolini
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  3. Carlo V. Bruschi
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Corresponding author

Correspondence to Carlo V. Bruschi.

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Communicated by: P. Hieter

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ESM 1

Colony PCR amplification of clones m1 and m2 of the diploid strain YF123 with primers: a = DUR3R/DURUP; b = SP1/DUR3R; c = ΔUR3′in/DURext; d = Sp3/ΔUR3′in; e = Sp2Fw/Sp2Rev; f = Sp2Fw/Sp2. The sequences of the primers are reported in ESM S2 (“Materials and methods”). The doublets (a, c, e; obtained with primers outside the point of insertion) indicate a size differences between the two chromosomes VIII due to the spacers insertion in the new diploid strain; the second amplification (b, d, f), which was obtained with one primer on the chromosome and the second primer within the spacer, confirms the insertion of the three spacers. The bands in lane d are very faint due to the great difference of annealing temperature of the primers SP3 and Dur3INS. To confirm the results, the amplification on Sp3 was also performed with primer sp3II (Table S2; not shown). The marker is the 1-kb plus ladder (Invitrogen). 60 × 45 mm (300 × 300 DPI) (GIF 10.1 KB)

High-resolution image file (TIF 1.12 MB)

ESM 2

Primers used in this work (from the 5′ to the 3′ end); the nucleotide position within the respective chromosome is reported in brackets (RTF 19.6 KB)

ESM 3

Colony PCR analysis of different kinds of transformants resulting from the KO experiment of DUR3. In a) the cassette replaced the DUR3 of chromosome VIII deriving from YPH250 (with Sp3). In b, the marker replaced DUR3 from Fas20, labeled with Sp1. In c, an ectopic integration is considered. In the picture model, a is characterized by a doublet of 866 and 750 bp (DURUP/DUR3R/k1) and by two bands of 920 and 315 nt (DURext/ΔUR3′in/k2); in b, the characteristic bands are of 980/620 (DURUP/DUR3R/k1) and 795/430 (DURext/ΔUR3′in/k2). In case of ectopic integration (c), the cassette is integrated in another locus of the genome; the PCR shows the same amplification sizes obtained also in the wild-type strain, which are: 750/620 (DURUP/DUR3R) and 430/315 (DURext/ΔUR3′in). 60 × 45 mm (300 × 300 DPI) (GIF 7.17 KB)

High-resolution image file (TIF 1.13 MB)

ESM 4

Colony PCR analysis of four transformants obtained with a ΔDUR3 cassette, showing the presence of three bands. In lanes 1, 2, and 3, a normal KO of DUR3 is presented. In lane 4, the amplification with three primers (k2, DURext, ΔUR3′in) leads to three bands. The small bands correspond to the two chromosomes VIII (with and without Sp3). The other end of the cassette is integrated ectopically into another chromosome as shown in the image on the left side. In half of the cases, a BIR-like event copies the chromosome VIII with Sp3. a = primers k1, DURUP, DUR3R; b = primers k2, DURext, ΔUR3′in. 60 × 45 mm (300 × 300 DPI) (GIF 4.43 KB)

High-resolution image file (TIF 1.12 MB)

ESM 5

Phenotypic analysis of several transformants and their comparison to the wild-type strain YF123. a = wild type before (left) and after (right) DAPI staining; b = the translocant; c, d, e, f = four different G418-resistant transformants indicating cell cycle and nuclei segregation defects. g = clone 44 after a few days of growth shows many dead cells and budding defects (GIF 336 KB)

High-resolution image file (TIF 305 KB)

ESM 6

Colony PCR amplification with primers DURUP/DUR3R/k1 upstream and sp2Fw/sp2Rev/k2 downstream (left and right of each numbered lane, respectively) of several G418-resistant clones resulting from the transformations with the 240-kb cassette. The size of the bands is reported in base pairs. Lanes 1, 7, and 10 show the pattern described in Fig. 3a, b, respectively. Lanes 3, 4, 5, 8, 9, 11, 12, 13, and 15 are examples of ectopic integrations. Lanes 2 and 6 show an ectopic pattern plus a band at approximately 1,059 bp in the integration downstream suggesting an upstream ectopic integration of the cassette and a putative BIR-like event downstream with a consequent partial trisomy of chromosome VIII. Lane 14 corresponds to a putative monosome for chromosome VIII (cl. 51) (GIF 168 KB)

High-resolution image file (TIF 104 KB)

ESM 7

Quantification of the expression of the cell-cycle-related and apoptotic genes CDC48, CLB1, and YCA1. Only CLB1 shows a significant decrease. The expression values are reported as the ratio between the translocant and the wild-type strain YF123. Three RT-PCR reactions were performed for each sample using different cDNAs. Each result is the average of nine independent readings. Each reading was related to the expression of an internal control in the same quantitative RT-PCR. The gene used as control is the constitutively expressed gene HSC82 (chr. XV). The standard deviation is reported on the top of each bar (GIF 121 KB).

High-resolution image file (TIF 67.2 KB)

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Tosato, V., Nicolini, C. & Bruschi, C.V. DNA bridging of yeast chromosomes VIII leads to near-reciprocal translocation and loss of heterozygosity with minor cellular defects. Chromosoma 118, 179–191 (2009). https://doi.org/10.1007/s00412-008-0187-z

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