Repair of Active and Silenced rDNA in Yeast

Meier, Andreas; Livingstone‐Zatchej, Magdalena; Thoma, Fritz

doi:10.1074/jbc.m110941200

Cited by 40 publications

(23 citation statements)

References 50 publications

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“…1A, lower panel [39]). Like NER (see description above), photolyase repair of CPDs in the yeast rDNA sequences is hindered by the presence of nucleosomes, and photoreactivation experiments confirmed the presence of CF and UAF at rDNA regulatory regions (32,33). In WT cells, NER is slow over the entire RNAPI promoter and downstream of the transcription initiation site up to nucleotide ϩ34 (Fig.…”

Section: Discussionmentioning

confidence: 96%

“…In contrast to human cells, yeast cells rapidly remove CPDs from rDNA (7,8). Furthermore, strand-specific repair was observed for rDNA of the rad7⌬ and rad16⌬ mutants (59), and TC-NER was measured in the active rDNA fraction of wild-type (WT) cells (7,10,33). Although NER was analyzed in total rDNA and important results were reported (14,15,59), a number of questions remain unanswered because those studies did not follow NERs separately in the active and inactive rDNA.…”

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confidence: 99%

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Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin

Tremblay

Teng

Paquette

et al. 2008

Molecular and Cellular Biology

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Nucleotide excision repair (NER) removes a plethora of DNA lesions. It is performed by a large multisubunit protein complex that finds and repairs damaged DNA in different chromatin contexts and nuclear domains. The nucleolus is the most transcriptionally active domain, and in yeast, transcription-coupled NER occurs in RNA polymerase I-transcribed genes (rDNA). Here we have analyzed the roles of two members of the xeroderma pigmentosum group C family of proteins, Rad4p and Rad34p, during NER in the active and inactive rDNA. We report that Rad4p is essential for repair in the intergenic spacer, the inactive rDNA coding region, and for strand-specific repair at the transcription initiation site, whereas Rad34p is not. Rad34p is necessary for transcription-coupled NER that starts about 40 nucleotides downstream of the transcription initiation site of the active rDNA, whereas Rad4p is not. Thus, although Rad4p and Rad34p share sequence homology, their roles in NER in the rDNA locus are almost entirely distinct and complementary. These results provide evidences that transcription-coupled NER and global genome NER participate in the removal of UV-induced DNA lesions from the transcribed strand of active rDNA. Furthermore, nonnucleosome rDNA is repaired faster than nucleosome rDNA, indicating that an open chromatin structure facilitates NER in vivo.

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Section: Discussionmentioning

confidence: 96%

mentioning

confidence: 99%

Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin

Tremblay

Teng

Paquette

et al. 2008

Molecular and Cellular Biology

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“…The CPD photolyases repair both active and inactive genes (39). Therefore, the enzyme should search for and repair CPD lesions without interfering with the transcriptional regulation.…”

Section: Table II Analyses Of the Interactions Between The Cpd Photolmentioning

confidence: 99%

Investigation of the Cyclobutane Pyrimidine Dimer (CPD) Photolyase DNA Recognition Mechanism by NMR Analyses

Torizawa

Ueda

Kuramitsu

et al. 2004

Journal of Biological Chemistry

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The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from 31 P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.Irradiation of DNA with ultraviolet (UV) light produces various damaged bases, leading to cellular transformation and cell death (1-3). One of the major products formed by UV irradiation is the cyclobutane pyrimidine dimer (CPD) 1 (Fig. 1), from the photo [2 ϩ 2] cycloaddition of the 5,6-double bond of two adjacent pyrimidine nucleotides. Organisms have a variety of enzymes playing crucial roles in repair-systems for damaged DNA, including nucleoside excision repair systems and photoreactivation (4). CPD photolyases, which function as members of the DNA repair systems, restore the CPD to normal pyrimidines by photoreactivation. To elucidate the mechanism of DNA repair by CPD photolyase, various biological and spectroscopic experiments have been performed. This enzyme has a flavin adenine dinucleotide (FAD) as an essential cofactor (5).The FAD is excited by light, and then transfers one electron to the CPD bases (6 -8). After the electron transfer, the cyclobutane ring splits and then one electron is transferred back to the FAD (9, 10). In order to understand the mechanism based on structural analyses, the crystal structures of the CPD photolyases from Escherichia coli, Anacystis nidulans, and Thermus thermophilus have been solved without the substrates, i.e. CPD-containing DNAs (11-13). The x-ray studies revealed that the enzymes share a similar global fold, which consists of an ␣/␤ domain and a helical domain. The helical domain is composed of clusters I and II, and a cavity is formed between the clusters, where the FAD is deeply buried. It has been suggested that the cavity is used for the CPD binding, because the asymmetric polarity of the cavity fits well with that of the CPD (11-15). Based upon the crystal structures without the substrates, two research groups have proposed computer models of the DNA-CPD photolyase complex, in which the relative orientations of the DNA chain are different from each other (11-15). However, no crystallographic or NMR structure information on the complexes is presently available.Here, we report NMR analyses of...

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“…This was first detected in human cells (47) and later in many other organisms, including yeast (42,48). Recently, transcription-coupled repair was also shown to occur in yeast ribosomal DNA transcribed by RNA-polymerase I (49,50).…”

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confidence: 99%

Repair of UV Lesions in Silenced Chromatin Provides in Vivo Evidence for a Compact Chromatin Structure

Livingstone‐Zatchej

Marcionelli

Möller

et al. 2003

Journal of Biological Chemistry

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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 sir3⌬ 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.Silencing refers to transcriptional inhibition characterized by the epigenetic formation of a repressive chromatin structure, frequently referred to as heterochromatin. In yeast Saccharomyces cerevisiae, silencing occurs at several genetic loci, including the cryptic mating-type loci (HML and HMR), the ribosomal DNA, and regions close to telomeres (1-4). Silencing of genes integrated in subtelomeric regions decreases with increasing distance from the telomere (telomere position effect) (5). In contrast to stable silencing at the HM loci, silencing of subtelomeric genes is variegated, resulting in stochastic patterns of repression of transcription (6 -9).Telomeric silencing depends on numerous proteins: three silent information regulators (Sir2, Sir3, and Sir4), histones, proteins required for chromatin assembly, proteins involved in telomere formation as well as enzymes that modify histones by deacetylation, ubiquitination, and methylation (5-7, 10 -16). Silencing is enforced by the proximity to a pool of concentrated Sir proteins, clustered telomeres, and perinuclear localization (17)(18)(19)(20). Perinuclear localization appears to be necessary, albeit not sufficient, for silencing (21). Current models propose a stepwise formation of telomeric heterochromatin (2, 4, 22). Rap1 binds to telomeric Rap1-binding sites and recruits Sir4. Sir4 recruits Sir3 and the NAD-dependent histone deacetylase Sir2. Sir2 deacetylates histone H4 at Lys-16, which allows Sir3 binding to nucleosomes. Sir3 recruits more Sir4 onto nucleosomes, and the process is repeated as the SIR complex spreads along chromatin away from the initiation site (23, 24). In addition, telomeric chromatin appears to be further stabilized by folding back of the telomere, which allow...

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Repair of Active and Silenced rDNA in Yeast

Cited by 40 publications

References 50 publications

Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin

Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin

Investigation of the Cyclobutane Pyrimidine Dimer (CPD) Photolyase DNA Recognition Mechanism by NMR Analyses

Repair of UV Lesions in Silenced Chromatin Provides in Vivo Evidence for a Compact Chromatin Structure

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