M Livingstone-Zatchej

ETH Zurich, Zürich, ZH, Switzerland

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Publications (16)79.19 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: In yeast, Rad7 and Rad16 are two proteins required for nucleotide excision repair (NER) of non-transcribed chromatin. They have roles in damage recognition, in the postincision steps of NER, and in ultraviolet-light-dependent histone H3 acetylation. Moreover, Rad16 is an ATP-ase of the SNF2 superfamily and therefore might facilitate chromatin repair by nucleosome remodelling. Here, we used yeast rad7 Delta rad16 Delta mutants and show that Rad7-Rad16 is also required for NER of UV-lesions in three functionally distinct nucleosome-free regions (NFRs), the promoter and 3'-end of the URA3 gene and the ARS1 origin of replication. Moreover, rapid repair of UV-lesions by photolyase confirmed that nucleosomes were absent and that neither UV-damage formation nor rad7 Delta rad16 Delta mutations altered chromatin accessibility in NFRs. The data are consistent with a role of Rad7-Rad16 in damage recognition and processing in absence of nucleosomes. An additional role in nucleosome remodelling is discussed.
    DNA Repair 06/2008; 7(5):734-43. · 4.27 Impact Factor
  • Chapter: Photolyase
    Magdalena Livingstone-Zatchej, Bernhard Suter, Fritz Thoma
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    ABSTRACT: Folding of DNA into nucleosomes and higher order chromatin structures restricts its accessibility to proteins and drugs. Hence, the location of histone octamers on the DNA sequence (nucleosome positions) as well as structural and dynamic properties of nucleosomes may play important roles in gene regulation, replication and DNA repair. Conventional approaches to characterize chromatin structure include (partial) purification of chromatin and characterization of DNA accessibility to nucleases (micrococcal nuclease, DNaseI) and chemical cleavage reagents (hydroxyl radicals, methidium propyl-EDTA-iron, copper phenanthroline). The cleavage sites are monitored using low- and high-resolution footprinting protocols. These techniques, however, expose the problem that chromatin extraction procedures could alter chromatin composition and structure, including nucleosome positioning. To investigate chromatin structures in vivo, alternative approaches are applied, such as expression of prokaryotic methyltransferases in Saccharomyces cerevisiae, the genome of which contains no endogenous detectable methylation (1,2). The sites of methylation can be measured after DNA isolation using methylation-sensitive restriction enzymes. This approach, however, requires expression of a foreign gene, and the resolution is restricted because of the sequence specificity of the methyltransferases.
    02/2008: pages 245-259;
  • Magdalena Livingstone-Zatchej, Rosanna Marcionelli, Kathrin Möller, Remko de Pril, Fritz Thoma
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    ABSTRACT: Genes positioned close to telomeres in yeast are silenced by a heterochromatin-like structure containing Sir proteins. To investigate whether silencing also affects DNA repair, we studied removal of UV lesions by photolyase and nucleotide excision repair (NER) in strains containing the URA3 gene inserted 2 kilobases from a telomere. URA3 was transcriptionally active in sir3delta mutants, partially silenced in SIR3 cells, or completely silenced by overexpression of SIR3 or deletion of RPD3. The active URA3 showed efficient repair by both pathways. Fast repair of the promoter and 3' end by photolyase reflected a non-nucleosomal structure. Partial silencing had no remarkable effect on photolyase but reduced repair by NER, indicating differential accessibility for the two repair reactions. Complete silencing inhibits NER and photolyase in the coding region as well as in the promoter and the 3'-end. Conventional nuclease footprinting analyses revealed subtle changes in the promoter proximal nucleosome under partially silenced conditions but a pronounced reorganization of chromatin extending over the whole gene in silenced chromatin. Thus, both repair systems are sensitive to chromatin changes associated with silencing and provide direct evidence for a compact structure of heterochromatin.
    Journal of Biological Chemistry 10/2003; 278(39):37471-9. · 4.65 Impact Factor
  • Andreas Meier, Magdalena Livingstone-Zatchej, Fritz Thoma
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    ABSTRACT: DNA repair by photolyase (photoreactivation) and nucleotide excision repair (NER) are the major pathways to remove UV-induced cyclobutane-pyrimidine dimers (CPDs). The nucleolus is a nuclear subcompartment containing the ribosomal RNA genes (rDNA) of which a fraction is transcribed by RNA polymerase I (RNAP-I), and the rest is silenced. Here yeast was used to investigate how photoreactivation and NER contribute to repair of active and inactive rDNA. Cells were irradiated with UV light and exposed to different repair conditions. Nuclei were isolated, and the active genes were separated from the inactive genes by restriction endonuclease digestion. CPDs were measured in total rDNA, in both fractions, and in the GAL10 gene. Repair in rDNA was as efficient as in GAL10 indicating that both pathways have unrestricted access to the nucleolus. Photoreactivation was much faster than NER and therefore was the predominant repair pathway. Active genes were faster repaired by photolyase than were silenced genes providing evidence for an open chromatin structure during repair. The transcribed strands of active genes, but not of inactive genes, were slightly faster repaired by NER providing evidence for transcription-coupled repair by RNAP-I. There was no pronounced inhibition of photoreactivation by RNAP-I in the transcribed strand, which is in contrast to genes transcribed by RNAP-II and suggests different stabilities of RNAP-I and RNAP-II stalled at CPDs.
    Journal of Biological Chemistry 05/2002; 277(14):11845-52. · 4.65 Impact Factor
  • Andreas Meier, Magdalena Livingstone-Zatchej, Fritz Thoma
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA repair by photolyase (photoreactivation) and nucleotide excision repair (NER) are the major pathways to remove UV-induced cyclobutane-pyrimidine dimers (CPDs). The nucleolus is a nuclear subcompartment containing the ribosomal RNA genes (rDNA) of which a fraction is transcribed by RNA polymerase I (RNAP-I), and the rest is silenced. Here yeast was used to investigate how photoreactivation and NER contribute to repair of active and inactive rDNA. Cells were irradiated with UV light and exposed to different repair conditions. Nuclei were isolated, and the active genes were separated from the inactive genes by restriction endonuclease digestion. CPDs were measured in total rDNA, in both fractions, and in the GAL10 gene. Repair in rDNA was as efficient as in GAL10 indicating that both pathways have unrestricted access to the nucleolus. Photoreactivation was much faster than NER and therefore was the predominant repair pathway. Active genes were faster repaired by photolyase than were silenced genes providing evidence for an open chromatin structure during repair. The transcribed strands of active genes, but not of inactive genes, were slightly faster repaired by NER providing evidence for transcription-coupled repair by RNAP-I. There was no pronounced inhibition of photoreactivation by RNAP-I in the transcribed strand, which is in contrast to genes transcribed by RNAP-II and suggests different stabilities of RNAP-I and RNAP-II stalled at CPDs.
    Journal of Biological Chemistry 04/2002; 277(14):11845-11852. · 4.65 Impact Factor
  • Source
    S Lopez, M Livingstone-Zatchej, S Jourdain, F Thoma, A Sentenac, M C Marsolier
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    ABSTRACT: Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6ADelta nhp6BDelta double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6ADelta nhp6BDelta cells at 37 degrees C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6ADelta nhp6BDelta cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxillary factors are required for the optimal transcription of at least some specific Pol III genes.
    Molecular and Cellular Biology 06/2001; 21(9):3096-104. · 5.37 Impact Factor
  • Source
    S Li, M Livingstone-Zatchej, R Gupta, M Meijer, F Thoma, M J Smerdon
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    ABSTRACT: Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure. Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium. Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region. However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time. Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes. Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction. There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site. Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction.
    Nucleic Acids Research 10/1999; 27(17):3610-20. · 8.28 Impact Factor
  • M Livingstone-Zatchej, B Suter, F Thoma
    Methods in molecular biology (Clifton, N.J.) 02/1999; 119:245-59. · 1.29 Impact Factor
  • B Suter, M Livingstone-Zatchej, F Thoma
    Methods in Enzymology 02/1999; 304:447-61. · 2.00 Impact Factor
  • M Livingstone-Zatchej, F Thoma
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    ABSTRACT: The structural and functional subunits of chromatin are nucleosome cores. In a nucleosome core 145 bp of DNA are coiled around the outer surface of an octamer of histone proteins which consists of a tetramer of 2(H3·H4) and two H2A·H2B dimers (1). DNA extending from the nucleosome core to the next nucleosome is called linker DNA. It varies in length from about 20 to 90 bp in different organisms or tissues or between individual nucleosomes. Histone H1 may be associated with linker DNA at the site where the DNA leaves the nucleosome. While core histones are well conserved and present in all eukaryotic organisms, H1 is most variable and may even be missing in some organisms such as yeast Saccharomyces cerevisiae. Nucleosomes are built from many different DNA sequences and may contain histone variants (subtypes) and modified histones (e.g., acetylated) which can affect their structural and dynamic properties (reviewed in ref. 2).
    Methods in molecular biology (Clifton, N.J.) 02/1999; 119:363-78. · 1.29 Impact Factor
  • Source
    M Livingstone-Zatchej, A Meier, B Suter, F Thoma
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    ABSTRACT: Yeast uses nucleotide excision repair (NER) and photolyase (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs) generated by ultraviolet light. In active genes, NER preferentially repairs the transcribed strand (TS). In contrast, we recently showed that photolyase preferentially repairs the non-transcribed strands (NTS) of the URA3 and HIS3 genes in minichromosomes. To test whether photoreactivation depends on transcription, repair of CPDs was investigated in the transcriptionally regulated GAL10 gene in a yeast strain deficient in NER [AMY3 (rad1Delta)]. In the active gene (cells grown in galactose), photoreactivation was fast in the NTS and slow in the TS demonstrating preferential repair of the NTS. In the inactive gene (cells grown in glucose), both strands were repaired at similar rates. This suggests that RNA polymerases II blocked at CPDs inhibit accessibility of CPDs to photolyase. In a strain in which both pathways are operational [W303-1a (RAD1)], no strand bias was observed either in the active or inactive gene, demonstrating that photoreactivation of the NTS compensates preferential repair of the TS by NER. Moreover, repair of the NTS was more quickly in the active gene than in the repressed gene indicating that transcription dependent disruption of chromatin facilitates repair of an active gene.
    Nucleic Acids Research 11/1997; 25(19):3795-800. · 8.28 Impact Factor
  • Source
    B Suter, M Livingstone-Zatchej, F Thoma
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    ABSTRACT: Yeast and many other organisms use nucleotide excision repair (NER) and photolyase in the presence of light (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs), a major class of DNA lesions generated by UV light. To study the role of photoreactivation at the chromatin level in vivo, we used yeast strains which contained minichromosomes (YRpTRURAP, YRpCS1) with well-characterized chromatin structures. The strains were either proficient (RAD1) or deficient (rad1 delta) in NER. In contrast to NER, photolyase rapidly repairs CPDs in non-nucleosomal regions, including promoters of active genes (URA3, HIS3, DED1) and in linker DNA between nucleosomes. CPDs in nucleosomes are much more resistant to photoreactivation. These results demonstrate a direct role of chromatin in modulation of a DNA repair process and an important role of photolyase in repair of damaged promoters with presumptive effects on gene regulation. In addition, photoreactivation provides an in vivo test for chromatin structure and stability. In active genes (URA3, HIS3), photolyase repairs the non-transcribed strand faster than the transcribed strand and can match fast removal of lesions from the transcribed strand by NER (transcription-coupled repair). Thus, the combination of both repair pathways ensures efficient repair of active genes.
    The EMBO Journal 05/1997; 16(8):2150-60. · 9.82 Impact Factor
  • Bernhard Suter, Magdalena Livingstone-Zatchej, Andreas Meier, Fritz Thoma
    Mutation Research-fundamental and Molecular Mechanisms of Mutagenesis - MUTAT RES-FUNDAM MOL MECH MUT. 01/1997; 379(1).
  • S Tanaka, M Livingstone-Zatchej, F Thoma
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    ABSTRACT: To characterize nucleosome structure and positioning in the chromosomal context, the chromatin structure of the whole URA3 gene was studied in the genome and in a minichromosome by testing the accessibility of DNA to micrococcal nuclease and DNase I. The cutting patterns and hence the chromatin structures were almost indistinguishable in the genome and in the minichromosomes. The only notable exception was enhanced cutting between nucleosomes U3/U4 and U4/U5 in the minichromosomes. The results demonstrate that there is no severe constraint acting from outside the URA3 gene in chromosomes and minichromosomes. While low-resolution mapping showed six regions with a positioned nucleosome (U1 to U6), each region resolved in a complex pattern consistent with multiple overlapping positions. Some regions (U1, U4, U5 and U6) showed multiple positions with a dominant rotational setting (DNase I pattern), while U2 showed positioning within 10 bp but with no defined rotational setting, demonstrating that nucleosome positions were not in phase and not coordinately regulated. Reduced DNase I cutting from about 50 bp form the 5' end towards 3' end was common to all nucleosome regions. This polarity has been observed on isolated core particles. The results demonstrate that the DNase I pattern observed in vitro indeed reflects a structural property of nucleosomes in the chromosomal context. It is emphasized that despite the local heterogeneity revealed by high-resolution mapping, the low-resolution map is a reasonably accurate representation of the chromatin structure.
    Journal of Molecular Biology 05/1996; 257(5):919-34. · 3.91 Impact Factor
  • M C Marsolier, S Tanaka, M Livingstone-Zatchej, M Grunstein, F Thoma, A Sentenac
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    ABSTRACT: Recent work has demonstrated a repressive effect of chromatin on the transcription of the yeast SNR6 gene in vitro. Here, we show the relations between chromatin structure and transcriptional activity of this gene in vivo. Analysis of the SNR6 locus by micrococcal nuclease digestion showed a protection of the TATA box, nuclease-sensitive sites around the A and B blocks, and arrays of positioned nucleosomes in the flanking regions. Analysis of a transcriptionally silent SNR6 mutant containing a 2-bp deletion in the B block showed a loss of TATA-protection and rearrangement or destabilization of nucleosomes in the flanking regions. Hence, SNR6 organizes the chromatin structure in the whole region in a manner dependent on its transcriptional state. Transcriptional analysis was performed by use of maxi-gene SNR6 constructs introduced into histone-mutated strains. Chromatin disruption induced by histone H4 depletion stimulated the transcription of promoter-deficient, but not of wild-type SNR6 genes, revealing a competition between the formation of nucleosomes and the assembly of Pol III transcription complexes that was much in favor of transcription factors. On the other hand, amino-terminal mutations in histone H3 or H4 had no effect (H4) or only a moderate stimulatory effect (H3) on the transcription of promoter-deficient SNR6 genes.
    Genes & Development 03/1995; 9(4):410-22. · 12.44 Impact Factor
  • Source
    S Tanaka, D Halter, M Livingstone-Zatchej, B Reszel, F Thoma
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    ABSTRACT: When the function of origins of replication in yeast was compromised by placing ARS sequences downstream of strong promoters, ARS activity might have been affected either by transcription or by an altered chromatin configuration induced by the construct. To distinguish between these possibilities, derivatives of the yeast TRP1ARS1 minichromosome were constructed that contained either the DED1 or the PET56 promoter firing against ARS1 (DEDARS and PETARS constructs). PETARS constructs transformed yeast at high frequencies and were maintained as minichromosomes consistent with efficient ARS1 function, but DEDARS constructs transformed at low frequencies and had to be rescued as minichromosomes by insertion of a second ARS (H4-ARS). Chromatin analysis revealed that the ARS1 regions in PETARS and H4-DEDARS constructs were indistinguishable from the ARS1 region of the host TRP1ARS1 circle showing a nuclease sensitive region flanked by a nucleosome. However, RNA-analysis in the ARS region showed high and low levels of transcripts in H4-DEDARS and PETARS, respectively. Transcription elongated through the A, B1, and B2 elements and ended in B3, the binding site for ABFI. We conclude that transcription through ARS1 and not an altered chromatin structure affected ARS activity in these constructs.
    Nucleic Acids Research 10/1994; 22(19):3904-10. · 8.28 Impact Factor