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.

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.

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.

High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents.

Intracellular metabolites and environmental agents continuously challenge the structural integrity of DNA. In the yeast Saccharomyces cerevisiae, the complete collection of open reading frame deletion mutants, in combination with powerful screening methods, allows for the comprehensive analyses of cellular responses to insult. We have developed a protocol to determine the sensitivity of growing yeast to DNA-damaging agents that is based on automatic measurements of the optical density of very small (100 microl) liquid cultures. This simple method is highly sensitive, provides quantifiable data and offers high-throughput screening capability. Starting with the treatment of cells with different doses of damaging agents, pre-prepared growing media containing 96-well plates are inoculated and cell population is automatically monitored every 10 min for 48 hours. With the aid of a multi-channel pipette, the sensitivity of a number of yeast strains to several concentrations of drug can be tested in triplicate in less then 4 hours.

Psoralen photocrosslinking, a tool to study the chromatin structure of RNA polymerase I--transcribed ribosomal genes.

The chromatin structure of RNA polymerase I--transcribed ribosomal DNA (rDNA) is well characterized. In most organisms, i.e., lower eukaryotes, plants, and animals, only a fraction of ribosomal genes are transcriptionally active. At the chromatin level inactive rDNA is assembled into arrays of nucleosomes, whereas transcriptionally active rDNA does not contain canonical nucleosomes. To separate inactive (nucleosomal) and active (non-nucleosomal) rDNA, the technique of psoralen photocrosslinking has been used successfully both in vitro and in vivo. In Saccharomyces cerevisiae, the structure of rDNA chromatin has been particularly well studied during transcription and during DNA replication. Thus, the yeast rDNA locus has become a good model system to study the interplay of all nuclear DNA processes and chromatin. In this review we focused on the studies of chromatin in ribosomal genes and how these results have helped to address the fundamental question: What is the structure of chromatin in the coding regions of genes?

Limited TTP supply affects telomere length regulation in a telomerase-independent fashion.

An adequate supply of nucleotides is essential for DNA replication and DNA repair. Moreover, inhibition of TTP synthesis can cause cell death by a poorly characterized mechanism called thymine-less death. In the yeast Saccharomyces cerevisiae, the genes encoding thymidylate synthetase (CDC21) and thymidylate kinase (CDC8) are both essential for de novo TTP synthesis. The effects of temperature-sensitive mutations in these genes have been characterized and, curiously, the phenotypes displayed by cells harboring them include shortened telomeric repeat tracts. This finding raised the possibility that the enzyme telomerase is very sensitive to TTP-pools. We tested this possibility in vivo by assessing telomerase-dependent extension in situations of lowered TTP supply. The results show that the above-mentioned short telomere phenotype is not a consequence of an inability of telomerase to elongate telomeres when TTP synthesis is impaired. Moreover, this telomere shortening was abolished in cells harboring a mutation in DNA polymerase alpha. Previously, this same mutation was shown to affect the coordination between conventional replication and telomerase-mediated extension. These results thus re-emphasize the importance of the interplay between conventional replication and telomerase-mediated addition of telomeric repeats in telomere replication.