Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.

Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage--including that induced by many anticancer drugs--results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events

Strains and relevant genotypes.

<p>Strains and relevant genotypes.</p

KP1019 induces a pre-anaphase cell cycle delay.

<p><b>A and B.</b> Cells arrested in the G1 phase of the cell cycle by mating pheromone were released in the absence (0 mg/mL KP1019) and presence (80 mg/mL KP1019) of drug. Pds1-myc <b>(A)</b> and budding index <b>(B)</b> were measured every 20 minutes after release from G1. <b>A.</b> Pds1-myc epitope tagged protein expression levels assayed as cells progress through the cell division cycle. Antibody against the Nop1 protein is used as loading control. Normalized Pds1 band intensities are represented below each blot. <b>B.</b> Budding index (% of unbudded cells in the population) of cells used to measure Pds1 levels as cells progress through the cell division cycle. <b>C.</b> Distance between SPB in large budded cells that are untreated (blue bar) or treated with 80 mg/mL KP1019 for three hours (red bar) were determined as described in Materials and Methods. The average of three replicates is presented with 2XSE error bar.</p

Nuclear Morphology in KP1019 treated cells.

<p><b>A.</b> Early log phase wild-type yeast were treated with 80ug/ml KP1019 for three hours and nuclear morphology was determined as described in Materials and Methods. Large budded cells were scored on the position of the nucleus relative to the bud neck. Categories are indicated on the X-axis, and include a single nuclei positioned near the bud neck and not migrating across the bud neck (single nucleus), cells with some protrusion of nucleus across bud neck (protrusion across bud neck), nuclei spanning the bud neck (bowtie), and two separated nuclei positioned in each cell body (separated). Example image of treated cells from each category is provided. <b>B.</b> Representative images from the cytological analysis used to obtain data in Parts A showing varying levels of nuclear migration across the bud neck. Red signal is Histone H2-mCherry indicating nuclear position. Green signal is Spc42-GFP indicating SPB position. Arrows indicate cells with short spindles; the white color indicates an untreated cell with a single nuclei, and the yellow color indicates treated cells showing migration of nuclei into the daughter cell. White asterisk show fully separated spindle in untreated cells at varying points in anaphase. Note more random orientation of SPB bodies relative to the longitudinal cell axis exampled in treated cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138085#pone.0138085.s001" target="_blank">S1 Fig</a>).</p

Dyn1 dependent KP1019 induced nuclear positions.

<p>Early log phase wild-type (MMY318-5A) and <i>dyn1</i> deletion (<i>dyn1Δ</i>) strains were treated with 80ug/ml KP1019 for three hours and assayed for budding index and nuclear position as described in Materials and Methods. <b>A.</b><i>DYN1</i> deletion does not impact KP1019 induced cell cycle arrest. Untreated samples are represented by blue (WT) and red (<i>dyn1Δ</i>) bars, while treated samples are represented by light blue (WT) and light red (<i>dyn1Δ</i>) bars. The average of three replicates is presented with 2XSE error bar. <b>B.</b> KP1019 induced nuclear migration across the bud neck is Dyn1 dependent. Large budded cells were scored for the position of the nucleus relative to the bud neck. Categories are indicated on the X-axis, and include a single nuclei positioned near the bud neck, but not migrating across the bud neck (bud neck), cells with some protrusion of nucleus across bud neck (protrusion across bud neck), a bowtie phenotype (nuclei spanning bud neck), and two separated nuclei positioned in each cell body (Nuclei separated). Untreated samples are represented by blue (WT) and red (<i>dyn1Δ</i>) bars, while treated samples are represented by light blue (WT) and light red (<i>dyn1Δ</i>) bars. The average of three replicates is presented with 2XSE error bar.</p

KP1019 induced <i>DUN1</i>-dependent expression of Hug1 protein and phosphorylation of Rad53.

<p>A. Early log phase wild-type (WT) and <i>dun1</i> deletion (<i>dun1Δ</i>) strains were treated with 80ug/ml KP1019 for three hours (+) or untreated (-) and assayed for Hug1 protein expression. Western blot analysis was carried out as described in Materials and Methods. Each sample was run in duplicate lanes and “*” indicates a non-specific band that serves as the loading control. B. WT cells were incubated with increasing concentrations of KP1019 for one hour and assayed for Rad53 protein phosphorylation. Western blots were carried out as described in Materials and Methods. The shift in position of Rad53 protein consistent with increased phosphorylation is indicated by Rad53-TAP-P, while unmodified Rad53 is indicated by Rad53-TAP.</p

Transcriptomic analysis of KP1019-induced changes in gene expression.

<p>Early log phase wild-type yeast were treated with either 80ug/ml (A and C) or 40ug/ml (B and D) KP1019 for three hours. Extraction of RNA and microarray analyses were carried out as described in Materials and Methods. Genes repressed (A and B) and induced (C and D) by KP109 were assembled into clusters using the MCL algorithm on the Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING; <a href="http://string-db.org/" target="_blank">http://string-db.org/</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138085#pone.0138085.ref044" target="_blank">44</a>]. Clusters were predicted with medium confidence and at an inflation level of 3 to allow for identification of a moderate number of clusters. Weak links and disconnected nodes are hidden for clarity. Node size indicates the presence (large nodes) or absence (small nodes) of structural data in the database. Within each cluster, the confidence of each inter-node connection is indicated by the width and intensity of the blue line. Graphs summarize biological process GO term analyses by DAVID. Redundant GO terms (like “dNTP metabolic process”) were omitted in the interest of clarity and brevity.</p