Modelling the spectral response of the Swift-XRT CCD camera: Experience learnt from in-flight calibration

(Abbreviated) We show that the XRT spectral response calibration was complicated by various energy offsets in photon counting (PC) and windowed timing (WT) modes related to the way the CCD is operated in orbit (variation in temperature during observations, contamination by optical light from the sunlit Earth and increase in charge transfer inefficiency). We describe how these effects can be corrected for in the ground processing software. We show that the low-energy response, the redistribution in spectra of absorbed sources, and the modelling of the line profile have been significantly improved since launch by introducing empirical corrections in our code when it was not possible to use a physical description. We note that the increase in CTI became noticeable in June 2006 (i.e. 14 months after launch), but the evidence of a more serious degradation in spectroscopic performance (line broadening and change in the low-energy response) due to large charge traps (i.e. faults in the Si crystal) became more significant after March 2007. We describe efforts to handle such changes in the spectral response. Finally, we show that the commanded increase in the substrate voltage from 0 to 6V on 2007 August 30 reduced the dark current, enabling the collection of useful science data at higher CCD temperature (up to -50C). We also briefly describe the plan to recalibrate the XRT response files at this new voltage.Comment: 27 pages, 29 figures (many in colour), accepted for publication in A&

Insights into the function of DNA repair factors MRN and ATM

DNA double strand breaks (DSB) are a particularly deleterious threat to genomic integrity throughout all domains of life. DSBs can cause chromosomal aberrations, tumorigenesis and cell death if left unre-paired and are caused by either endogenous or exogenous sources. Cells rely on efficient detection, repair and response upon occurrence of DSBs. In eukaryotes, DSBs are mostly repaired by either end joining pathways or homologous recombination (HR). HR, in contrast to the end joining pathways, en-ables error-free DSB repair in presence of a template sister chromatid. The Mre11-Rad50-Nbs1 (MRN) complex recognizes and tethers DNA ends, even if they are obstructed by proteins to initiate HR. In order to respond to DSBs, the MRN complex recruits and activates the signaling kinase Ataxia-telangiectasia mutated (ATM), that belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. Activated ATM in turn initiates the cellular DNA damage response (DDR). Mre11 and Rad50 are highly conserved and form a topology-specific, ATP-dependent nuclease complex that pro-cesses DNA ends but leaves genomic DNA intact. The eukaryote specific Nbs1 subunit finetunes MRN’s endonuclease activity by providing interaction with proteins (e.g. CtIP). Apart from its nucleo-lytic activity, MRN has a scaffolding function that promotes DNA end tethering, repair foci formation and possibly signal amplification. Although the complex has been studied for more than two decades, a model that integrates both MRN’s enzymatic and scaffolding functions has not yet been established. In the first part of the thesis, such a model was elaborated by combining both structural and biochemical data from this and previ-ous studies. A cryo-electron microscopy (cryo-EM) structure of the Chaetomium thermophilum (Ct)MRN catalytic head domain in its ATPγS-bound state not only clarifies its atomic architecture but also reveals how a core part of Nbs1 stabilizes and possibly locks the Mre11 dimer. In this structure significant parts of the Rad50 coiled-coils were resolved in a rod configuration, stabilized by several interaction points. A previously uncharacterized C-terminal Mre11 domain, denoted bridge could fur-ther stabilize the rod configuration. The rod configuration and the bridge domain restrict access to the Rad50 DNA binding site. Biochemical analysis revealed the Rad50 DNA binding site is extremely specific for DNA ends. However, an additional, eukaryote-specific DNA binding site at the C-terminus of Mre11 enables binding to internal DNA. The Rad50 coiled-coil domains are linked at the apex via a zinc hook dimerization motif to form a large proteinaceous ring/rod. Cryo-EM data and crystal structures ex-plained how two MRN complexes can tether DNA ends via dimerization of these apical domains. In vivo assays indicate that mutation of the apex tethering element negatively impacts DSB repair. Mutations in DDR pathways allow cancer cells to cope with increased replication and genotoxic stress. For this reason, proteins involved in DDR were described to be promising targets in cancer therapy. Due to its central role in DSB induced DDR, ATM is an auspicious target for drug development. Howev-er, lack of ATM high-resolution structures, as well as atomic details of small molecule inhibitor binding modalities hampered the application of structure-based drug design. In the second part of the thesis, the binding modalities of two ATP-competitive ATM-inhibitors were described. This project was a col-laborative work with Merck KGaA, that provided a novel inhibitor (M4076) with improved pharmacoki-netics. Comparison of the inhibitor-bound kinase active sites with the likewise resolved ATPγS-bound active site explains the high affinities that were determined in biochemical assays. Superposition and sequence alignment of the ATM kinase active site with other PIKK active sites enables to rationalize the molecular reasons for selectivity. In biochemical assays, IC50 values of the inhibitors for ATM, PIKKs and CHK2 showed high selectivity towards ATM. The binding of the inhibitors stabilized the N-terminal solenoid domain of ATM, this enabled the generation of a high-resolution structure of the entire ATM protein. The quality of the map allowed the identification of two zinc binding sites that possibly stabi-lize loops and the generation of a near-complete ATM structure. Taken together, the structural data provides the framework for structure-based ATM inhibitor design and allows mapping of cancer muta-tion as well as functionally important protein interaction sites

Three installations: a thesis chronicle

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Ride the Tide: Observing CRISPR/Cas9 genome editing by the numbers

Targeted genome editing has become a powerful genetic tool for modification of DNA sequences in their natural chromosomal context. CRISPR RNA-guided nucleases have recently emerged as an efficient targeted editing tool for multiple organisms. Hereby a double strand break is introduced at a targeted DNA site. During DNA repair genomic alterations are introduced which can change the function of the DNA code. However, our understanding of how CRISPR works is incomplete and it is still hard to predict the CRISPR activity at the precise target sites. The highly ordered structure of the eukaryotic genome may play a role in this. The organization of the genome is controlled by dynamic changes of DNA methylation, histone modification, histone variant incorporation and nucleosome remodelling. The influence of nuclear organization and chromatin structure on transcription is reasonably well known, but we are just beginning to understand its effect on genome editing by CRISP

Functional Characterisation of a RECQL4 Mutation in Rothmund-Thomson Syndrome

Germline mutations affecting the RECQL4 DNA helicase cause Type II Rothmund-Thomson syndrome (RTS), a human disease characterised by defects in skeletal development and predisposition to specific types of cancer, including osteosarcoma (OS). RECQL4 has been implicated in multiple cellular functions that mediate accurate DNA replication and repair. How germline RECQL4 mutations associated with Type II RTS affect these functions to cause disease remains unclear, in part due to the paucity of appropriate cellular models. In this work, CRISPR/Cas9 gene editing was used to generate cell lines containing a prevalent RTS patient RECQL4 mutation, the “Mut-2” c.2269C>T. The resulting Mut-2 clones exhibited greatly reduced RECQL4 protein levels, similar to decreases observed in RTS patient cells. Unexpectedly, the major effect of this predicted nonsense mutation was the upregulation of the use of an alternative splice site in exon 14 which skipped the premature stop codon and resulted in the deletion of 66 amino acids in the RECQL4 ATPase domain. Despite the lower overall RECQL4 expression, single cell clones bearing the Mut-2 mutation showed mostly normal cell cycle distribution with a slight increase in population doubling times. When challenged with various DNA damaging agents, these Mut-2 clones exhibited increased sensitivities to DNA alkylators and topoisomerase inhibitors, and mild sensitivities to DNA crosslinkers and PARP inhibitors, a sensitivity profile suggestive of defects in DNA double-strand break (DSB) repair. When further assayed using flow cytometric GFP reporters, the Mut-2 clones showed decreased DNA DSB repair capacities in the homologous recombination (HR) and microhomology mediated end joining (MMEJ) pathways, providing evidence that RECQL4 disruption impacted replication-specific DNA DSB repair in particular. Additional RECQL4 reconstitution studies confirmed that the decreased HR repair was a result of structural changes to RECQL4 due to the Mut-2 mutation. Finally, the formation of RAD51 foci—a commonly used marker of HR function—in the Mut-2 clones post-DNA DSB induction was investigated. Surprisingly, upon DNA DSB challenge, all Mut-2 clones were as proficient at forming RAD51 foci as parental HEK293. This suggested that the RECQL4 Mut-2 mutation disrupted its function further downstream in the HR pathway than had been previously reported. The work presented in this dissertation is a novel approach to studying the effects of clinical RTS RECQL4 mutations. These studies have illuminated mechanisms of RECQL4 disruption in Type II RTS as well as the roles of the RECQL4 helicase in cellular DNA damage repair. Because about 30% of Type II RTS patients are diagnosed with osteosarcoma, a common and deadly primary malignancy of the bone, the results presented here could shed new light on potential mechanisms underlying osteosarcoma tumour development and ultimately suggest new avenues and strategies for targeted clinical intervention.NIH OxCam Progra

Risk of well integrity failure due sustained casing pressure

Sustained casing pressure (SCP) is considered a well integrity problem. The approach of this study is to look at SCP as environmental risk due hydrocarbon release. Currently, the risk is qualified by the value of surface pressure (Pcsg) that may cause failure of casing head. However, the resulting rate of gas emission to the atmosphere is not considered. Also not considered is a possibility of breaching the casing shoe due transmission of Pcsg downhole. The objective of this study is to develop methods for maximum possible air emission rates (MER) and risk of subsurface well integrity failure due SCP. Mathematical models and software are developed for computing MER, casing shoe strength (CSS) determined by leak-off test (LOT), and casing shoe pressure load resulting from SCP (SCPd). The models are used to find controlling parameters, identify the best and least-desirable scenarios, and assess environmental risk. It is concluded that emission potential of SCP wells with high wellhead pressure (Pcsg) can be quite small. The CSS model study reveals the importance of data recorded from LOT; particularly the time after circulation was stopped – the non-circulation time (∆ts). Ignoring ∆ts would result in underestimation of the ultimate CSS. The error is caused by the cumulative effect of thermally induced rock stresses, which strongly depend on ∆ts. The study displayed SCPd being controlled by the annular fluid properties which are subject to change in long time through mud aging; and mostly being overestimated. Comparison of surface versus subsurface failure scenarios yielded cases where the casing shoe demonstrates more restrictive failure criterion (CSS) than the burst rating of wellhead (MAWOP). Risk of casing shoe breaching (RK) is quantified using the CSS and SCPd models and application of risk analysis technique (QRA). The CSS distribution followed log-normal trend due the effect of ∆ts, while the SCPd distribution maybe of various shapes dependent on the annular fluid size and properties that are not well known. Possible scenarios of casing shoe breaching are statistically tested as a hypothesis of two means. The study produced engrossingly variant outcomes, RK changing from 1 to 80 percent