Regulation of Stability and Copy Number of Tandem Repeats in <i>Saccharomyces cerevisiae</i>

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, I used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. I developed powerful, highly sensitive assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating rDNA copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of the CUP1 array, I showed that instability at both tandem arrays can be induced by DNA replication stress and altered transcription of the locus. Importantly, while changes in instability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and, importantly, it restricts transcription-induced amplification of the CUP1 array. My work suggests that the modulation of replication and transcription is a simple, reversible strategy to alter instability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the ubiquity of DNA replication, transcription, and chromatin marks like histone acetylation, the mechanisms I have characterized could significantly advance our understanding of the behavior of other tandem repeats and their role in shaping eukaryotic genomes

DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number

Ribosomal DNA instability and genome adaptability

Ribosomes are large, multi-subunit ribonucleoprotein complexes, essential for protein synthesis. To meet the high cellular demand for ribosomes, all eukaryotes have numerous copies of ribosomal DNA (rDNA) genes that encode ribosomal RNA (rRNA), usually far in excess of the requirement for ribosome biogenesis. In all eukaryotes studied, rDNA genes are arranged in one or more clusters of tandem repeats localized to nucleoli. The tandem arrangement of repeats, combined with the high rates of transcription at the rDNA loci, and the difficulty of replicating repetitive sequences make the rDNA inherently unstable and particularly susceptible to large variations in repeat copy number. Despite mounting evidence suggesting extra-ribosomal functions of the rDNA, its repetitive nature has excluded it from traditional sequencing-based studies. However, more recently, several studies have revealed the unique potential of the rDNA to act as a "canary in the coalmine," being particularly sensitive to genomic stresses and acting as a source of adaptive response. Here, we review evidence uncovering mechanisms of regulation of instability and copy number variation at the rDNA and their role in adaptation to the environment, which could serve to understand the basic principles governing the behavior of other tandem repeats and their role in shaping the genome

Pengaruh Fasilitas Kerja, Komunikasi Kerja, Disiplin Kerja Terhadap Kinerja Karyawan Pada PT. Multi Mitra Baruna

Penelitian ini bertujuan untuk mengetahui dan menganalisis pengaruh lingkungan kerja, komunikasi kerja, dan disiplin kerja terhadap kinerja karyawan PT. Fenomena fasilitas perusahaan yang kurang memadai, keterbatasan anggaran, komunikasi yang kurang baik dengan rekan kerja, dimana karyawan cenderung membentuk kelompok-kelompok tersendiri, serta tingkat kedisiplinan dan kesadaran diri karyawan dan atasan dalam mematuhi peraturan yang terindikasi rendah menjadi latar belakang dilakukannya penelitian ini. Penelitian ini bersifat kuantitatif. Dengan jumlah sampel karyawan sebanyak 45 orang, metode pengambilan sampel yang digunakan adalah metode sampling jenuh. Teknik analisis data yang digunakan adalah regresi linier berganda. Hasil penelitian menunjukkan bahwa fasilitas kerja berpengaruh positif dan signifikan secara statistik terhadap kinerja karyawan di PT. Multi Mitra Baruna, begitu juga dengan komunikasi kerja dan disiplin kerja. Selain itu, pengaruh fasilitas kerja, komunikasi kerja, dan disiplin kerja terhadap kinerja karyawan PT.Multi Mitra Baruna

Contraction of the rDNA array promotes timely completion of DNA replication and cell cycle progression.

<p>Wild-type <i>GAL-POL1</i> cells were subcultured in medium containing high or low levels of galactose for ~75 generations to generate 3 independent isolates each with normal or low rDNA copy number (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.s006" target="_blank">S2 Table</a>). (A) Representative DNA content profiles over time are shown for asynchronous cultures of isolates with normal (i) and low (ii) rDNA copy number following inoculation into the indicated medium which determines high or low levels of DNA polymerase α. (B) Fraction of cells in S-phase in each of the 4 conditions in (A). Error bars indicate standard deviation based on 3 independent isolates. Statistical significance of differences between fraction of cells in S-phase in high and low levels of DNA polymerase α was calculated using a standard 2-tailed t-test. *—p<0.05, ****—p<0.0001. (C) Increased rARS firing in nicotinamide exacerbates growth defects under conditions of DNA replication stress.</p

Low rDNA copy number confers advantage under DNA replication stress.

<p>(A) 3 independent isolates each with normal or low rDNA copy number were generated by subcultuing wild-type or <i>fob1Δ GAL-POL1</i> cells in medium containing high or low galactose (high or low levels of Pol1 respectively) for ~50–75 generations (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.s006" target="_blank">S2 Table</a>) and 5-fold serial dilutions spotted on to medium with DNA replication stress (low Pol1). (B) 10-fold serial dilutions of wild-type and <i>fob1Δ</i> cells (~200–250 rDNA copies) along with cells having 20–110 rDNA copies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.ref003" target="_blank">3</a>] were spotted on medium containing hydroxyurea (HU). rDNA copy number was confirmed by ddPCR. Error bars represent standard deviation for each individual reaction.</p

Screen for essential genes that maintain rDNA copy number.

<p>(A) Distribution of rDNA copy number across the yeast ts mutant collection. 175 and 113 strains had significantly lower (<70 copies) and higher copy number (>119 copies) respectively (p<0.05). (B) -log₁₀ transformed FDR q-values for significantly enriched (p<0.001) GO terms (sorted in order of increasing p-values from top to bottom) in low copy number hits.</p

Design and validation of a ddPCR assay for rDNA copy number measurement.

<p>(A) Targets (red bars) within the rDNA and single copy reference (<i>TUB1</i>) loci in yeast. (B) rDNA copy number in 3 independent isolates each of 2 different wild-type laboratory yeast strains, BY4741 and W303, and 4 isogenic strains with varying rDNA copy number generated by Ide et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.ref003" target="_blank">3</a>]. (C) rDNA copy number in 8 technical replicates each of BY4741 and W303. (D) rDNA copy number in 3 independent isolates each of mutants with expanded rDNA arrays. Error bars represent standard deviation for each individual reaction.</p