Distinctive Participation of Transcription-Coupled and Global Genome Nucleotide Excision Repair of Pyrimidine Dimers in the Transcribed Strand of Yeast rRNA Genes.

UV light causes the formation of pyrimidine dimers (PDs). Transcription-coupled (TC) nucleotide excision repair (NER) and global genome (GG) NER remove PDs from the transcribed strand (TS) of active genes and the inactive genome, respectively. TC-NER is triggered by elongating RNA polymerases that are blocked at PDs. The yeast rRNA genes are densely loaded with RNA polymerase-I. After UV irradiation, their density increases at the 5'-end of the gene, which results from continuous transcription initiation, followed by elongation and pausing/release at the first encountered PD, from the transcription start site. RNA polymerase-I posed at downstream PDs are released from the TS and are replaced by nucleosomes. Consequently, discrete chromatin structures are formed in the damaged transcribed rRNA genes. Singular assignation of the two NER sub-pathways could therefore be required to eliminate PDs from the TS. To advance our understanding of NER in the dynamic structure of transcribed chromatin, we investigated the repair of PDs at nucleotide resolution in separate rRNA gene coding regions. In the TS, the TC-NER efficiency reflected the density of RNA polymerase-I, and PDs were removed faster in the 5'-end than in the 3'-end of the gene. GG-NER removed PDs from the TS where RNA polymerase-I was transiently replaced by a nucleosome. The two NER sub-pathways inversely participated to remove PDs from the TS. In the non-TS of both nucleosome and non-nucleosome rRNA gene coding regions, GG-NER was solely responsible to remove UV-induced DNA lesions.

Transcription of ncRNAs promotes repair of UV induced DNA lesions in Saccharomyces cerevisiae subtelomeres.

Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure. UV induced photoproducts are repaired faster in the gene transcribed strands than in the non-transcribed strands or in transcriptionally inactive regions of the genome. This specificity of NER is known as transcription-coupled repair (TCR). The discovery of pervasive non-coding RNA transcription (ncRNA) advocates for ubiquitous contribution of TCR to the repair of UV photoproducts, beyond the repair of active gene-transcribed strands. Chromatin rules transcription, and telomeres form a complex structure of proteins that silences nearby engineered ectopic genes. The essential protective function of telomeres also includes preventing unwanted repair of double-strand breaks. Thus, telomeres were thought to be transcriptionally inert, but more recently, ncRNA transcription was found to initiate in subtelomeric regions. On the other hand, induced DNA lesions like the UV photoproducts must be recognized and repaired also at the ends of chromosomes. In this study, repair of UV induced DNA lesions was analyzed in the subtelomeric regions of budding yeast. The T4-endonuclease V nicking-activity at cyclobutene pyrimidine dimer (CPD) sites was exploited to monitor CPD formation and repair. The presence of two photoproducts, CPDs and pyrimidine (6,4)-pyrimidones (6-4PPs), was verified by the effective and precise blockage of Taq DNA polymerase at these sites. The results indicate that UV photoproducts in silenced heterochromatin are slowly repaired, but that ncRNA transcription enhances NER throughout one subtelomeric element, called Y', and in distinct short segments of the second, more conserved element, called X. Therefore, ncRNA-transcription dependent TCR assists global genome repair to remove CPDs and 6-4PPs from subtelomeric DNA.

The Efficiency of Global Genome-Nucleotide Excision Repair is Linked to the Fraction of Open rRNA Gene Chromatin, in Yeast.

The yeast rDNA locus is a suitable model to study nucleotide excision repair (NER) in chromatin. A portion of rRNA genes is transcribed and largely depleted of nucleosomes, the remaining genes are not transcribed and folded in nucleosomes. In G1-arrested cells, most rRNA genes do not have nucleosomes. TC-NER removes UV-induced DNA lesions from the transcribed strand of active genes. GG-NER is less efficient and removes DNA lesions from the nontranscribed strand of active genes and from the inactive genome. Different from mammalian cells, in yeast, the rRNA gene-transcribed strand is repaired by RNA polymerase-I-dependent TC-NER. The opposite nontranscribed strand is repaired faster than both strands of inactive rRNA genes. In log-phase cells, RNA polymerase-I are dislodged from the damaged transcribed strand and partially replaced by nucleosomes. Contrary to log-phase cells, in G1-phase cells few, if any, histones are deposited on the open rRNA genes during NER. In this study, we compared GG-NER efficiency in the rRNA gene coding region: without nucleosomes, partially loaded or wholly loaded with nucleosomes. The results indicate that in log-phase cells histones obstruct GG-NER, whereas in G1-phase cells GG-NER is as efficient as TC-NER.

Analyses of rRNA gene chromatin in cell cycle arrested Saccharomyces cerevisiae cells.

The existence of two chromatin structures in the rDNA locus was previously demonstrated for a large variety of organisms, ranging from yeast to human. In yeast there are about 150-200 rRNA genes organized in tandem repeats. Almost half of them are transcribed and largely depleted of nucleosomes (active/open), the other half is not transcribed and is assembled in regular arrays of nucleosomes (inactive/closed). It is proposed that RNA polymerase-I (RNAPI) transcription-elongation removes nucleosomes from closed rRNA genes (opening), and that soon after DNA replication there is deposition of nucleosomes on the open rRNA genes (closing). In G1 arrested cells, nearly all rRNA genes are depleted of nucleosomes, but most of them are not transcribed (inactive/open). In relation to the research article by Charton et al. (Mutat. Res.), the data presented here are on the hydroxyurea concentration-dependent inhibition of yeast culture growth, on cell cycle arrest before completion of genome replication, and on the opening of rRNA gene chromatin. As comparison, data are presented for yeast arrested in the G1-phase of the cell cycle by the pheromone α-factor.

In yeast cells arrested at the early S-phase by hydroxyurea, rRNA gene promoters and chromatin are poised for transcription while rRNA synthesis is compromised.

Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase that is used as a chemotherapeutic agent to treat a number of chronic diseases. Addition of HU to cell cultures causes reduction of the dNTP cellular pool below levels that are required for DNA replication. This trigger dividing cells to arrest in early S-phase of the cell cycle. Cell division hinges on ribosome biogenesis, which is tightly regulated by rRNA synthesis. Remarkably, HU represses the expression of some genes the products of which are required for rRNA maturation. To gain more information on the cellular response to HU, we employed the yeast Saccharomyces cerevisiae as model organism and analyzed the changing aspects of closed to open forms of rRNA gene chromatin during cell cycle arrest, the arrangement of RNA polymerase-I (RNAPI) on the open genes, the presence of RNAPI transcription-factors, transcription and rRNA maturation. The rRNA gene chromatin structure was analyzed by psoralen crosslinking and the distribution of RNAPI was investigated by chromatin endogenous cleavage. In HU arrested cells nearly all rRNA genes were in the open form of chromatin, but only a portion of them was engaged with RNAPI. Analyses by chromatin immuno-precipitation confirmed that the overall formation of transcription pre-initiation complexes remained unchanged, suggesting that the onset of rRNA gene activation was not significantly affected by HU. Moreover, the in vitro transcription run-on assay indicated that RNAPI retained most of its transcription elongation activity. However, in HU treated cells, we found that: (1) RNAPI accumulated next to the 5'-end of rRNA genes; (2) considerably less rRNA filaments were observed in electron micrographs of rDNA transcription units; and (3) rRNA maturation was compromised. It is established that HU inhibition of ribonucleotide reductase holds back DNA replication. This study indicates a hitherto unexplored cellular response to HU, namely altered rRNA synthesis, which could participate to hamper cell division.

Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu-Sir4 interaction.

Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y'-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y'-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu-Sir4 interaction, but Sir2 and Sir3 independent.

RNA Polymerase-I-Dependent Transcription-coupled Nucleotide Excision Repair of UV-Induced DNA Lesions at Transcription Termination Sites, in Saccharomyces cerevisiae.

If not repaired, ultraviolet light-induced DNA damage can lead to genome instability. Nucleotide excision repair (NER) of UV photoproducts is generally fast in the coding region of genes, where RNA polymerase-II (RNAP2) arrest at damage sites and trigger transcription-coupled NER (TC-NER). In Saccharomyces cerevisiae, there is RNA polymerase-I (RNAP1)-dependent TC-NER, but this process remains elusive. Therefore, we wished to characterize TC-NER efficiency in different regions of the rDNA locus: where RNAP1 are present at high density and start transcription elongation, where the elongation rate is slow, and in the transcription terminator where RNAP1 pause, accumulate and then are released. The Rpa12 subunit of RNAP1 and the Nsi1 protein participate in transcription termination, and NER efficiency was compared between wild type and cells lacking Rpa12 or Nsi1. The presence of RNAP1 was determined by chromatin endogenous cleavage and chromatin immunoprecipitation, and repair was followed at nucleotide precision with an assay that is based on the blockage of Taq polymerase by UV photoproducts. We describe that TC-NER, which is modulated by the RNAP1 level and elongation rate, ends at the 35S rRNA gene transcription termination site.

Immuno-capture of UVDE generated 3'-OH ends at UV photoproducts.

A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.

Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae.

The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.

Repair of UV induced DNA lesions in ribosomal gene chromatin and the role of "Odd" RNA polymerases (I and III).

In fast growing eukaryotic cells, a subset of rRNA genes are transcribed at very high rates by RNA polymerase I (RNAPI). Nuclease digestion-assays and psoralen crosslinking have shown that they are open; that is, largely devoid of nucleosomes. In the yeast Saccharomyces cerevisae, nucleotide excision repair (NER) and photolyase remove UV photoproducts faster from open rRNA genes than from closed and nucleosome-loaded inactive rRNA genes. After UV irradiation, rRNA transcription declines because RNAPI halt at UV photoproducts and are then displaced from the transcribed strand. When the DNA lesion is quickly recognized by NER, it is the sub-pathway transcription-coupled TC-NER that removes the UV photoproduct. If dislodged RNAPI are replaced by nucleosomes before NER recognizes the lesion, then it is the sub-pathway global genome GG-NER that removes the UV photoproducts from the transcribed strand. Also, GG-NER maneuvers in the non-transcribed strand of open genes and in both strands of closed rRNA genes. After repair, transcription resumes and elongating RNAPI reopen the rRNA gene. In higher eukaryotes, NER in rRNA genes is inefficient and there is no evidence for TC-NER. Moreover, TC-NER does not occur in RNA polymerase III transcribed genes of both, yeast and human fibroblast.

UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes.

The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5'-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin.

Chromatin endogenous cleavage and psoralen crosslinking assays to analyze rRNA gene chromatin in vivo.

In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

UV-induced DNA damage and DNA repair in ribosomal genes chromatin.

Cyclobutane pyrimidine dimers (CPDs) and (6,4) pyrimidine-pyrimidone dimers are the major DNA lesions (or photoproducts) induced by ultraviolet light and are removed by the nucleotide excision repair (NER) pathway. If not repaired, DNA damage can lead to genome instability. The genome is organized into nuclear domains with distinct functions and chromatin structures. Although studies on NER in all chromosomal contexts are important to understand the mechanisms of genome maintenance, we focused on NER in the nucleolus. The attractive feature of the rDNA locus is its chromatin structure; not all rRNA genes are transcribed and both active (no nucleosomes) and inactive (nucleosomes) rRNA genes coexist in the nucleolus. These characteristics allow for direct comparison of NER in two very different chromatin structures. Yeast is used as a model system and the methods employed are as follows: nuclei isolation, restriction enzyme digestion of chromatin to release active rRNA genes, psoralen cross-linking, T4-endonuclease-V enzyme to detect CPDs and CPDs repair over relatively large stretches of DNA, and primer extension to follow DNA damage and repair at nucleotide level. Using this approach, we have shown that NER is faster in nonnucleosomes vs. nucleosomes rDNA, that the formation of CPDs promotes changes in the active rDNA chromatin, and that NER is coupled to rRNA genes transcription.

Differential participation of homologous recombination and nucleotide excision repair in yeast survival to ultraviolet light radiation.

AIMS: The purpose of this research was to assess the ultraviolet light (UV) phenotype of yeast sirDelta cells vs. WT cells, and to determine whether de-silenced chromatin or the intrinsic pseudoploidy of sirDelta mutants contributes to their response to UV. Additional aims were to study the participation of HR and NER in promoting UV survival during the cell cycle, and to define the extent of the co-participation for both repair pathways. MAIN METHODS: The sensitivity of yeast Saccharomyces cerevisiae to UV light was determined using a method based on automatic measurements of optical densities of very small (100mul) liquid cell cultures. KEY FINDINGS: We show that pseudo-diploidy of sirDelta strains promotes resistance to UV irradiation and that HR is the main mechanism that is responsible for this phenotype. In addition, HR together with GG-NER renders cells in the G2-phase of the cell cycle more resistant to UV irradiation than cells in the G1-phase, which underscore the importance of HR when two copies of the chromosomes are present. Nevertheless, in asynchronously growing cells NER is the main repair pathway that responds to UV induced DNA damage. SIGNIFICANCE: This study provides detailed and quantitative information on the co-participation of HR and NER in UV survival of yeast cells.

Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivo.

The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA.

Complementary roles of yeast Rad4p and Rad34p in nucleotide excision repair of active and inactive rRNA gene chromatin.

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.

Deletion of Rnt1p alters the proportion of open versus closed rRNA gene repeats in yeast.

In Saccharomyces cerevisiae, the double-stranded-RNA-specific RNase III (Rnt1p) is required for the processing of pre-rRNA and coprecipitates with transcriptionally active rRNA gene repeats. Here we show that Rnt1p physically interacts with RNA polymerase I (RNAPI) and its deletion decreases the transcription of the rRNA gene and increases the number of rRNA genes with an open chromatin structure. In contrast, depletion of ribosomal proteins or factors that impair RNAPI termination did not increase the number of open rRNA gene repeats, suggesting that changes in the ratio of open and closed rRNA gene chromatin is not due to a nonspecific response to ribosome depletion or impaired termination. The results demonstrate that defects in pre-rRNA processing can influence the chromatin structure of the rRNA gene arrays and reveal links among the rRNA gene chromatin, transcription, and processing.

A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures.

The sensitivity of yeast Saccharomyces cerevisiae to DNA damaging agents is better represented when cells are grown in liquid media than on solid plates. However, systematic assessment of several strains that are grown in different conditions is a cumbersome undertaking. We report an assay to determine cell growth based on automatic measurements of optical densities of very small (100 microl) liquid cell cultures. Furthermore, an algorithm was elaborated to analyze large data files obtained from the cell growth curves, which are described by the growth rate--that starts at zero and accelerates to the maximal rate (mu(m))--and by the lag time (lambda). Cell dilution spot test for colony formation on solid media and the growth curve assay were used in parallel to analyze the phenotypes of cells after treatments with three different classes of DNA damaging agents (methyl methanesulfonate, bleomycin, and ultraviolet light). In these experiments the survival of the WT (wild type) and a number of DNA repair-deficient strains were compared. The results show that only the cell growth curve assay could uncover subtle phenotypes when WT cells, or mutant strains that are only weakly affected in DNA repair proficiency, were treated with low doses of cytotoxic compounds. The growth curve assay was also applied to establish whether histone acetyltransferases and deacetylases affect the resistance of yeast cells to UV irradiation. Out of 20 strains tested the sir2delta and rpd3delta cells were found to be more resistant than the WT, while gcn5delta and spt10delta cells were found to be more sensitive. This new protocol is sensitive, provides quantifiable data, offers increased screening capability and speed compared to the colony formation test.