Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

Nonhomologous end-joining (NHEJ) pathways repair DNA double-strand breaks (DSBs) in eukaryotes and many prokaryotes, although it is not reported to operate in the third domain of life, archaea. Here, we describe a complete NHEJ complex, consisting of DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku from a mesophillic archaeon, Methanocella paludicola (Mpa). Mpa Lig has limited DNA nick-sealing activity but is efficient in ligating nicks containing a 3′ ribonucleotide. Mpa Pol preferentially incorporates nucleoside triphosphates onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3′ phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3′ monoribonucleotide. These proteins, together with the DNA end-binding protein Ku, form a functional NHEJ break-repair apparatus that is highly homologous to the bacterial complex. Although the major roles of Pol and Lig in break repair have been reported, PE’s function in NHEJ has remained obscure. We establish that PE is required for ribonucleolytic resection of RNA intermediates at annealed DSBs. Polymerase-catalyzed strand-displacement synthesis on DNA gaps can result in the formation of nonligatable NHEJ intermediates. The function of PE in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides or phosphates from 3′ ends of annealed DSBs to configure the termini for ligation. Thus, PE prevents the accumulation of abortive genotoxic DNA intermediates arising from strand displacement synthesis that otherwise would be refractory to repair

Structural and functional characterisation of the Nonhomologous End-­‐Joining proteins of the archaeon Methanocella Paludicola

Maintenance of the genome is essential for life to prosper. Regular insults to the genome are sustained by all cellular life and can foster genetic instability if left unrepaired. The most lethal genetic damage is a double strand break (DSB), the cleavage of the phosphate backbone on both strands of the DNA double helix. Two main pathways exist which provide mechanisms for coping with DSBs; precise repair utilising the identical sister chromatid as a template to recreate the broken segment (homologous recombination; HR), and direct fusion of the broken ends in the absence of an intact template (nonhomologous end joining; NHEJ). NHEJ was first characterised in eukaryotes, and an analogous system has been found to exist in bacteria during the past decade. The bacterial NHEJ pathway is composed of four key proteins; the DNA end binding Ku homodimer, a DNA Ligase, a DNA polymerase and a phosphoesterase (PE). The first results chapter of this thesis details the identification of an orthologous set of proteins in the archaeon Methanocella paludicola, and their subsequent isolation and characterisation. The second results chapter expands on the individual activities of the proteins by combining them, and asserting the ability of archaeal NHEJ to join discontinuous ends in vitro. The role of the PE has been unclear in the bacterial system, but in vitro assays described here suggest that the enzyme plays a role in processing NHEJ intermediates formed by the NHEJ polymerase. The PE is found to optimise repair intermediates for ligation, and to reverse potentially genotoxic DNA strand displacements. The final results chapter investigates the structural aspects of the archaeal NHEJ enzymes. Together these studies establish a functional NHEJ system in an archaeon for the first time, and expand our knowledge of the bacterial system by proposing a standard model of archaeo--‐prokaryotic NHEJ

Molecular basis for DNA strand displacement by NHEJ repair polymerases

The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair

Dosimetric Comparison of Treatment Techniques: Brachytherapy, Intensity- Modulated Radiation Therapy, and Proton Beam in Partial Breast Irradiation

Purpose: To perform a dosimetric comparison of 3 accelerated partial breast irradiation techniques: catheter-based brachytherapy (BT), intensity-modulated radiation therapy (IMRT), and proton beam therapy (PBT). Patients and Methods: Twelve patients with left-sided breast cancer treated with SAVI (Strut-Adjusted Volume Implant) were selected in this study. The original BT plans were compared with optimum plans using IMRT and PBT for 34 Gy (RBE) with 1.1 RBE in 10 fractions using identical parameters for target and organs at risk. Results: Significant reduction in maximum dose to the ipsilateral breast was observed with PBT and IMRT (mean 108.58% [PBT] versus 107.78% [IMRT] versus 2194.43% [BT], P = .001 for both PBT and IMRT compared to BT). The mean dose to the heart was 0%, 1.38%, and 3.85%, for PBT, IMRT, and BT, respectively (P < .001 and P = .026). The chest wall mean dose was 10.07%, 14.65%, and 29.44% for PBT, IMRT, and BT, respectively (P = .001 and .013 compared to BT). The PBT was superior in reducing the mean ipsilateral lung dose (mean 0.04% versus 2.13% versus 5.4%, P = .025 and P < .001). There was no statistically significant difference in the maximum dose to the ipsilateral lung, chest wall, 3-mm skin rind or in the mean ipsilateral breast V50% among the 3 techniques (P = .168, .405, .067, and .780, respectively). PBT exhibited the greatest mean dose homogeneity index of 4.75 compared to 7.18 for IMRT (P = .001) and 195.82 for BT (P < .001). All techniques resulted in similar dose conformality (P = .143). Conclusion: This study confirms the dosimetric feasibility of PBT and IMRT to lower dose to organs at risk while still maintaining high target dose conformality. Though the results of this comparison are promising, continued clinical research is needed to better define the role of PBT and IMRT in the accelerated partial breast irradiation treatment of early-stage breast cancer

Plug-and-play inference for disease dynamics: measles in large and small populations as a case study

Statistical inference for mechanistic models of partially observed dynamic systems is an active area of research. Most existing inference methods place substantial restrictions upon the form of models that can be fitted and hence upon the nature of the scientific hypotheses that can be entertained and the data that can be used to evaluate them. In contrast, the so-called plug-and-play methods require only simulations from a model and are thus free of such restrictions. We show the utility of the plug-and-play approach in the context of an investigation of measles transmission dynamics. Our novel methodology enables us to ask and answer questions that previous analyses have been unable to address. Specifically, we demonstrate that plug-and-play methods permit the development of a modelling and inference framework applicable to data from both large and small populations. We thereby obtain novel insights into the nature of heterogeneity in mixing and comment on the importance of including extra-demographic stochasticity as a means of dealing with environmental stochasticity and model misspecification. Our approach is readily applicable to many other epidemiological and ecological systems

LSST Science Book, Version 2.0

A survey that can cover the sky in optical bands over wide fields to faint magnitudes with a fast cadence will enable many of the exciting science opportunities of the next decade. The Large Synoptic Survey Telescope (LSST) will have an effective aperture of 6.7 meters and an imaging camera with field of view of 9.6 deg^2, and will be devoted to a ten-year imaging survey over 20,000 deg^2 south of +15 deg. Each pointing will be imaged 2000 times with fifteen second exposures in six broad bands from 0.35 to 1.1 microns, to a total point-source depth of r~27.5. The LSST Science Book describes the basic parameters of the LSST hardware, software, and observing plans. The book discusses educational and outreach opportunities, then goes on to describe a broad range of science that LSST will revolutionize: mapping the inner and outer Solar System, stellar populations in the Milky Way and nearby galaxies, the structure of the Milky Way disk and halo and other objects in the Local Volume, transient and variable objects both at low and high redshift, and the properties of normal and active galaxies at low and high redshift. It then turns to far-field cosmological topics, exploring properties of supernovae to z~1, strong and weak lensing, the large-scale distribution of galaxies and baryon oscillations, and how these different probes may be combined to constrain cosmological models and the physics of dark energy.Comment: 596 pages. Also available at full resolution at http://www.lsst.org/lsst/sciboo

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2

TDP2 is a 5’-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II. TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for HTS-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanised’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2

Large-scale ozone and aerosol distributions, air mass characteristics, and ozone fluxes over the western Pacific Ocean in late winter/early spring

Large‐scale measurements of ozone (O3) and aerosol distributions were made from the NASA DC‐8 aircraft during the Transport and Chemical Evolution over the Pacific (TRACE‐P) field experiment conducted in February–April 2001. Remote measurements were made with an airborne lidar to provide O3 and multiple‐wavelength aerosol backscatter profiles from near the surface to above the tropopause along the flight track. In situ measurements of O3, aerosols, and a wide range of trace gases were made onboard the DC‐8. Five‐day backward trajectories were used in conjunction with the O3 and aerosol distributions on each flight to indicate the possible origin of observed air masses, such as from biomass burning regions, continental pollution, desert regions, and oceanic regions. Average latitudinal O3 and aerosol scattering ratio distributions were derived from all flights west of 150°E, and these distributions showed the average latitude and altitude dependence of different dynamical and chemical processes in determining the atmospheric composition over the western Pacific. TRACE‐P (TP) showed an increase in the average latitudinal distributions of both O3 and aerosols compared to PEM‐West B (PWB), which was conducted in February–March 1994. O3, aerosol, and potential vorticity levels were used to identify nine air mass types and quantify their frequency of occurrence as a function of altitude. This paper discusses the characteristics of the different air mass types encountered during TP and compares them to PWB. These results confirmed that most of the O3 increase in TP was due to photochemistry. The average latitudinal eastward O3 flux in the western Pacific during TP was found to peak near 32°N with a total average O3 flux between 14 and 46°N of 5.2 Tg/day. The eastward total CO flux was calculated to be 2.2 Tg‐C/day with ∼6% estimated from Asia. The Asian flux of CO2 and CH4 was estimated at 4.9 and 0.06 Tg‐C/day