Multi-BRCT Domain Protein Brc1 Links Rhp18/Rad18 and γH2A To Maintain Genome Stability during S Phase.

DNA replication involves the inherent risk of genome instability, since replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during the S phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phosphohistone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1 to 4 of Brc1 have remained obscure. Here, we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1 overexpression suppression of smc6-74, a mutation that impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings, we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

High-resolution mutational profiling suggests the genetic validity of glioblastoma patient-derived pre-clinical models.

Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development, which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently, representative DNA biomarkers become equally important in pre-clinical studies. However, it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here, we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres, adherent cell cultures, and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes, and new actionable mutations such as the loss of PIK3R1, and reveals clear patient-to-patient differences. In contrast, for each patient, we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly, we observe ∼96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles, we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests, at a remarkable resolution, the genome-wide conservation of a patient's tumor genetics in various pre-clinical models, and therefore supports their use for the development and testing of personalized targeted therapies.

γH2A-binding protein Brc1 affects centromere function in fission yeast.

The coordinated replication and transcription of pericentromeric repeats enable RNA interference (RNAi)-mediated transmission of pericentromeric heterochromatin in fission yeast, which is essential for the proper function of centromeres. Rad3/ATR kinase phosphorylates histone H2A on serine-128/-129 to create γH2A in pericentromeric heterochromatin during S phase, which recruits Brc1 through its breast cancer gene 1 protein (BRCA1) C-terminal (BRCT) domains. Brc1 prevents the collapse of stalled replication forks; however, it is unknown whether this activity influences centromere function. Here, we show that Brc1 localizes in pericentromeric heterochromatin during S phase, where it enhances Clr4/Suv39-mediated H3 lysine-9 dimethylation (H3K9me2) and gene silencing. Loss of Brc1 increases sensitivity to the microtubule-destabilizing drug thiabendazole (TBZ) and increases chromosome missegregation in the presence of TBZ. Brc1 retains significant function even when it cannot bind γH2A. However, elimination of the serine-121 site on histone H2A, a target of Bub1 spindle assembly checkpoint kinase, sensitizes γH2A-deficient and brc1Δ cells to replication stress and microtubule destabilization. Collective results suggest that Brc1-mediated stabilization of stalled replication forks is necessary for fully efficient transmission of pericentromeric heterochromatin, which is required for accurate chromosome segregation during mitosis.

Replication fork collapse and genome instability in a deoxycytidylate deaminase mutant.

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.

Detection of low prevalence somatic mutations in solid tumors with ultra-deep targeted sequencing.

Ultra-deep targeted sequencing (UDT-Seq) can identify subclonal somatic mutations in tumor samples. Early assays' limited breadth and depth restrict their clinical utility. Here, we target 71 kb of mutational hotspots in 42 cancer genes. We present novel methods enhancing both laboratory workflow and mutation detection. We evaluate UDT-Seq true sensitivity and specificity (> 94% and > 99%, respectively) for low prevalence mutations in a mixing experiment and demonstrate its utility using six tumor samples. With an improved performance when run on the Illumina Miseq, the UDT-Seq assay is well suited for clinical applications to guide therapy and study clonal selection in heterogeneous samples.

Rad3 decorates critical chromosomal domains with gammaH2A to protect genome integrity during S-Phase in fission yeast.

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (gammaH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that gammaH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

Morphological control and assembly of zinc oxide using a biotemplate.

Zinc oxide is a wide band gap material that has significant applications in photovoltaics, piezoelectrics and optoelectronics. Traditionally, ZnO has been synthesized using high temperatures and harsh reaction conditions. Recently, benign reaction conditions have been used to synthesize ZnO using amine and citrate additives. In this study, peptide phage display was performed to identify a peptide, termed Z1, that binds to and directs the growth of ZnO hexagonal nanocrystals. By altering the concentration of Z1 peptide, the ZnO nanocrystal morphology can be tailored. Additionally, Z1 peptide was used to direct the growth of ZnO structures on free-standing silk films. The results presented here demonstrate the utility of peptides in controlling the structure and deposition of ZnO.

Protection of telomeres by a conserved Stn1-Ten1 complex.

Telomeres are specialized chromatin structures that protect chromosome ends. Critical among telomere proteins are those that bind the telomeric single-strand DNA (ssDNA) overhangs. These proteins are thought to differ among eukaryotes. Three interacting proteins (Cdc13, Stn1, and Ten1) associate with the telomeric overhang in budding yeast, a single protein known as Pot1 (protection of telomeres-1) performs this function in fission yeast, and a two-subunit complex consisting of POT1 and TPP1 associates with telomeric ssDNA in humans. Cdc13 and Pot1 have related oligonucleotide/oligosaccharide-binding fold (OB-fold) domains that bind the telomeric ssDNA overhang. Here we show that Schizosaccharomyces pombe has Stn1- and Ten1-like proteins that are essential for chromosome end protection. Stn1 orthologs exist in all species that have Pot1, whereas Ten1-like proteins can be found in all fungi. Fission yeast Stn1 and Ten1 localize at telomeres in a manner that correlates with the length of the ssDNA overhang, suggesting that they specifically associate with the telomeric ssDNA. Unlike in budding yeast, in which Cdc13, Stn1, and Ten1 all interact, fission yeast Stn1 and Ten1 associate with each other, but not with Pot1. Our findings suggest that two separate protein complexes are required for chromosome end protection in fission yeast. Structural profiling studies detect OB-fold domains in Stn1 and Ten1 orthologs, indicating that protection of telomeres by multiple proteins with OB-fold domains is conserved in eukaryotic evolution.

Characterization of microbial contamination in United States Air Force aviation fuel tanks.

Bacteria and fungi, isolated from United States Air Force (USAF) aviation fuel samples, were identified by gas chromatograph fatty acid methyl ester (GC-FAME) profiling and 16S or 18S rRNA gene sequencing. Thirty-six samples from 11 geographically separated USAF bases were collected. At each base, an above-ground storage tank, a refueling truck, and an aircraft wing tank were sampled at the lowest sample point, or sump, to investigate microbial diversity and dispersion within the fuel distribution chain. Twelve genera, including four Bacillus species and two Staphylococcus species, were isolated and identified. Bacillus licheniformis, the most prevalent organism isolated, was found at seven of the 11 bases. Of the organisms identified, Bacillus sp., Micrococcus luteus, Sphinogmonas sp., Staphylococcus sp., and the fungus Aureobasidium pullulans have previously been isolated from aviation fuel samples. The bacteria Pantoea ananatis, Arthrobacter sp., Alcaligenes sp., Kocuria rhizophilia, Leucobacter komagatae, Dietza sp., and the fungus Discophaerina fagi have not been previously reported in USAF aviation fuel. Only at two bases were the same organisms isolated from all three sample points in the fuel supply distribution chain. Isolation of previously undocumented organisms suggests either, changes in aviation fuel microbial community in response to changes in aviation fuel composition, additives and biocide use, or simply, improvements in isolation and identification techniques.

Cellular internalization and targeting of semiconductor quantum dots.

Peptide-mediated internalization and organelle targeting of quantum dots.