MPE•Fe(II) Footprinting: Drug Binding Sites on Native DNA

Many small molecules, such as antibiotics used in cancer chemotherapy, are believed to execute their therapeutic action through binding to the DNA template and impeding the progress of transcription and replication. While it is possible by spectroscopic means to determine the overall affinities and stoichiometries of these small molecule/DNA interactions, the exact locations and sizes of these sites of interaction are not known. The analogous question of protein: DNA binding specificities has been answered through the use of DNase I footprinting. This technique combines DNase I cleavage of protein protected DNA fragments and Maxam-Gilbert sequence determination methods, relying on the relatively low specificity of DNase I in a partial digestion and the ability of DNA-bound proteins to prevent phosphodiester bond hydrolysis between the base pairs they cover. Reported within is a direct technique, MPE•Fe(II) footprinting, which allows the determination of the preferred binding sites of several small molecules on heterogeneous double helical DNAs. Methidiumpropyl EDTA [MPE], in the presence of ferrous ion and oxygen efficiently creates single-strand breaks in double helical DNA and with significantly lower sequence specificity than DNase I. Utilizing MPE•Fe(II) as a small synthetic scissor one is capable of footprinting the preferred locations and binding site sizes of small molecules bound on native DNA. The small molecules actinomycin D, chromomycin A3, distamycin A, echinomycin, mithramycin, netropsin, and olivomycin have been shown to demonstrate sequence specific MPE•Fe(II) cleavage inhibition, thus allowing a determination of their preferred binding sites and site sizes. Actinomycin D was found to have a minimum site size of 3 base pairs and an absolute guanine requirement in its binding site. Distamycin A and netropsin demonstrated equivalent binding specificities, preferring contiguous A+T rich regions of 5 base pairs in length. Chromomycin A3, mithramycin, and olivomycin all shared similar binding specificities, demonstrating typically 3 base pair binding site sizes and preferring a 5'-GC-3' sequence within. Echinomycin protected a minimum of 4 base pairs; nearly all sites containing a central 5'-CG-3' sequence with 5'-CCGG-3' being favored. All footprints demonstrated an opposite strand asymmetry with overprotection on the 3' end. From a collection of their preferred binding sites, binding models for these small molecules have been derived. MPE•Fe(II) footprinting has been compared with DNase I footprinting in their ability to determine the binding specificities of both small molecules and proteins. While DNase I exhibits a slightly greater sensitivity, MPE•Fe(II) footprinting provided greater resolving capacity and importantly more precisely defined the binding locations and sizes of small molecule: DNA complexes. Both methods provided similar information in the case of a sequence-specific DNA binding protein, lac repressor. Investigations were also undertaken to study the effects of altered DNA conformation (Z-form DNA; binding cooperativity of different small molecules) on the interactions of small molecules with native DNA.</p

Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973

Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism’s transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5′–AACnAACGTTnGTT–3′ that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms

Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA

One of the first steps towards elucidating the biological function of a putative transcriptional regulator is to ascertain its preferred DNA-binding sequences. This may be rapidly and effectively achieved through the application of a combinatorial approach, one involving very large numbers of randomized oligonucleotides and reiterative selection and amplification steps to enrich for high-affinity nucleic acid-binding sequences. Previously, we had developed the novel combinatorial approach Restriction Endonuclease Protection, Selection and Amplification (REPSA), which relies not on the physical separation of ligand-nucleic acid complexes but instead selects on the basis of ligand-dependent inhibition of enzymatic template inactivation, specifically cleavage by type IIS restriction endonucleases. Thus, no prior knowledge of the ligand is required for REPSA, making it more amenable for discovery purposes. Here we describe using REPSA, massively parallel sequencing, and bioinformatics to identify the preferred DNA-binding sites for the transcriptional regulator SbtR, encoded by the TTHA0167 gene from the model extreme thermophile Thermus thermophilus HB8. From the resulting position weight matrix, we can identify multiple operons potentially regulated by SbtR and postulate a biological role for this protein in regulating extracellular transport processes. Our study provides a proof-of-concept for the application of REPSA for the identification of preferred DNA-binding sites for orphan transcriptional regulators and a first step towards determining their possible biological roles

Cleanup worker exposures to hazardous chemicals at a former nuclear weapons plant : piloting of an exposure surveillance system

Cleanup of former U.S. Department of Energy (DOE) nuclear weapons production facilities involves potential exposures to various hazardous chemicals. We have collaboratively developed and piloted an exposure database and surveillance system for cleanup worker hazardous chemical exposure data with a cleanup contractor at the Rocky Flats Environmental Technology Site (RFETS). A unique system feature is the incorporation of a 34-category work task-coding scheme. This report presents an overview of the data captured by this system during development and piloting from March 1995 through August 1998. All air samples collected were entered into the system. Of the 859 breathing zone samples collected, 103 unique employees and 39 unique compounds were represented. Breathing zone exposure levels were usually low (86% of breathing zone samples were below analytical limits of detection). The use of respirators and other exposure controls was high (87 and 88%, respectively). Occasional high-level excursions did occur. Detailed quantitative summaries are provided for the six most monitored compounds: asbestos, beryllium, carbon tetrachloride, chromium, lead, and methylene chloride. Task and job title data were successfully collected for most samples, and showed specific cleanup activities by pipe fitters to be the most commonly represented in the database. Importantly, these results demonstrate the feasibility of the implementation of integrated exposure database and surveillance systems by practicing industrial hygienists employed in industry as well as the preventive potential and research uses of such systems. This exposure database and surveillance system--the central features of which are applicable in any industrial work setting--has enabled one of the first systematic quantitative characterizations of DOE cleanup worker exposures to hazardous chemicals

Development of an exposure database and surveillance system for use by practicing OSH professionals

This report summarizes the development of an occupational exposure database and surveillance system for use by health and safety professionals at Rocky Flats Environmental Technology Site (RFETS), a former nuclear weapons production facility. The site itself is currently in the cleanup stage with work expected to continue into 2006. The system was developed with the intent of helping health and safety personnel not only to manage and analyze exposure monitoring data, but also to identify exposure determinants during the highly variable cleanup work. Utilizing a series of focused meetings with health and safety personnel from two of the major contractors at RFETS, core data elements were established. These data elements were selected based on their utility for analysis and identification of exposure determinants. A task-based coding scheme was employed to better define the highly variable work. The coding scheme consisted of a two-tiered hierarchical list with a total of 34 possible combinations of work type and task. The data elements were incorporated into a Microsoft Access database with built-in data entry features to both promote consistency and limit entry choices to enable stratified analyses. In designing the system, emphasis was placed on the ability of end users to perform complex analyses and multiparameter queries to identify trends in their exposure data. A very flexible and user-friendly report generator was built into the system. This report generator allowed users to perform multiparameter queries using an intuitive system with very little training. In addition, a number of automated graphical analyses were built into the system, including ex posure levels by any combination of building, date, employee, job classification, type of contaminant, work type or task, exposure levels over time, exposure levels relative to the permissible exposure limit (PELS), and distributions of exposure levels. Both of these interfaces, allow the user to \u27\u27drill down\u27\u27 or gradually narrow query criteria to identify specific exposure determinants. A number of other industrial hygiene processes were automated by the use of this database. Exposure calculations were coded into the system to allow automatic calculation of time-weighted averages and sample volumes. In addition, a table containing all the PELs and other relevant occupational exposure limits was built into the system to allow automatic comparisons with the current standards. Finally, the process of generating reports for employee notification was automated. The implementation of this system demonstrates that an integrated database system can save time for a practicing hygienist as well as provide useful and more importantly, timely information to guide primary prevention efforts

Recent FUSE Observations of Diffuse O VI Emission from the Interstellar Medium

We present new results from our survey of diffuse O VI-emitting gas in the interstellar medium with the Far Ultraviolet Spectroscopic Explorer (FUSE). Background observations obtained since 2005 have yielded eleven new O VI detections of 3-sigma significance, and archival searches have revealed two more. An additional 15 sight lines yield interesting upper limits. Combined with previous results, these observations reveal the large-scale structure of the O VI-bearing gas in the quadrant of the sky centered on the Magellanic Clouds. The most prominent feature is a layer of low-velocity O VI emission extending more than 70 degrees from the Galactic plane. At low latitudes (|b| < 30 degrees), the emission comes from narrow, high-density conductive interfaces in the local ISM. At high latitudes, the emission is from extended, low-density regions in the Galactic halo. We also detect O VI emission from the interface region of the Magellanic System, a structure recently identified from H I observations. These are the first detections of emission from high-ionization species in the Magellanic System outside of the Clouds themselves.Comment: 18 pages, 11 Postscript figures. To appear in Ap

ORFEUS-II Far-Ultraviolet Observations of 3C273: 1. Interstellar and Intergalactic Absorption Lines

We present the first intermediate-resolution (lambda / 3000) spectrum of the bright quasi-stellar object 3C273 at wavelengths between 900 and 1200 A. Observations were performed with the Berkeley spectrograph aboard the ORFEUS-SPAS II mission. We detect Lyman beta counterparts to previously-identified intergalactic Lyman-alpha features at cz = 19900, 1600, and 1000 km/s; counterparts to other putative Lyman-alpha clouds along the sight line are below our detection limit. The strengths of the two very low redshift Lyman-beta features, which are believed to arise in Virgo intracluster gas, exceed preflight expectations, suggesting that the previous determination of the cloud parameters may underestimate the true column densities. A curve-of-growth analysis sets a minimum H I column density of 4 E14/cm^2 for the 1600 km/s cloud. We find marginally significant evidence for Galactic H_2 along the sight line, with a total column density of about 1 E15/cm^2. We detect the stronger interstellar O VI doublet member unambiguously; the weaker member is blended with other features. If the Doppler b value for O VI is comparable to that determined for N V then the O VI column density is 7 +/- 2 E14/cm^2, significantly above the only previous estimate. The O VI / N V ratio is about 10, consistent with the low end of the range observed in the disk. Additional interstellar species detected for the first time toward 3C273 (at modest statistical significance) include P II, Fe III, Ar I, and S III.Comment: LaTeX file, 11 pages, 4 encapsulated PostScript figures. Uses aaspp4.sty and astrobib.sty. (Astrobib is available from http://www.stsci.edu/software/TeX.html .) The ORFEUS telescope is described at http://sag-www.ssl.berkeley.edu/orfeus/ . To appear in ApJ (Letters

Development and piloting of an exposure database and surveillance system for DOE cleanup operations

An industrial hygiene exposure database and surveillance system was developed in partnership between National Institute for Occupational Safety and Health (NIOSH)-funded independent investigators and practicing industrial hygienists at the Rocky Flats Environmental Technology Site (RFETS) in Golden, Colo. RFETS is a former U.S. Department of Energy nuclear weapons plant that is now in cleanup phase. This project is presented as a case study in the development of an exposure database and surveillance system in terms that are generalizable to most other industries and work contexts. Steps include gaining organizational support; defining system purpose and scope; defining database elements and coding; planning practical and efficient analysis strategies; incorporating reporting capabilities; and anticipating communication strategies that maximize the probability that surveillance findings will feed back to preventive applications. For each of these topics, the authors describe both general considerations as well as the specific choices made for this system. An important feature of the system is a two-tier task-coding scheme comprising 33 categories of task groups. Examples of grouped analyses of exposure data captured during the system pilot period demonstrate applications to exposure control, medical surveillance, and other preventive measures. Reprinted by permission of the publisher

Integrating workplace exposure databases for occupational medicine services and epidemiologic studies at a former nuclear weapons facility

We outline methods for integrating epidemiologic and industrial hygiene data systems for the purpose of exposure estimation, exposure surveillance, worker notification, and occupational medicine practice. We present examples of these methods from our work at the Rocky Flats Plant?a former nuclear weapons facility that fabricated plutonium triggers for nuclear weapons and is now being decontaminated and decommissioned. The weapons production processes exposed workers to plutonium, gamma photons, neutrons, beryllium, asbestos, and several hazardous chemical agents, including chlorinated hydrocarbons and heavy metals. We developed a job exposure matrix (JEM) for estimating exposures to 10 chemical agents in 20 buildings for 120 different job categories over a production history spanning 34 years. With the JEM, we estimated lifetime chemical exposures for about 12,000 of the 16,000 former production workers. We show how the JEM database is used to estimate cumulative exposures over different time periods for epidemiological studies and to provide notification and determine eligibility for a medical screening program developed for former workers. We designed an industrial hygiene data system for maintaining exposure data for current cleanup workers. We describe how this system can be used for exposure surveillance and linked with the JEM and databases on radiation doses to develop lifetime exposure histories and to determine appropriate medical monitoring tests for current cleanup workers. We also present time-line-based graphical methods for reviewing and correcting exposure estimates and reporting them to individual workers

Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer

BACKGROUND: Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. METHODS: Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. RESULTS: Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts. CONCLUSIONS: Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors