Fair Pavilion

V diplomové práci je vypracován návrh a statický posudek nosné ocelové konstrukce veletržního pavilonu o celkových půdorysných rozměrech 47x66m s maximální výškou 15,4m. Dispozice je navrhnuta v souladu s architektonickými požadavky. Konstrukce je uvažována pro oblast Brno. Návrh zastřešení je vypracován ve dvou variantách jejichž rozměry jsou shodné. První variantu tvoří dvoulodní halový objekt, příhradový parabolický vazník s vetknutými sloupy. Druhou variantu tvoří příhradový rám konstrukce. Ve statickém výpočtu jsou navrženy a posouzeny nosné prvky jako jsou vaznice, vazník, příčná a podélná ztužidla, průvlak a stropnice, štítové sloupy a kotvení. V závěru je zpracováno porovnání obou variant. Součástí práce je výkresová dokumentace skládající se u dispozičního výkresu, výrobního výkresu vazníku, výkresu detailů a kotevním plánem.Diploma thesis is developed the design and static assessment supporting steel structure Exhibition Hall of the overall ground dimensions 47x66m with a maximum height of 15.4 meters. Layout is designed in accordance with architectural requirements. The design is considered for Brno´s area. The proposal of roof system is designed in two versions whose dimensions are identical. The first option consists of a two-aisle hall object, parabolic truss girder, pin supported on fixed columns. The second variant also two-aisle building with truss frame structure assessed in static calculation. At the end of the thesis, comparison of both variants is elaborated. Design documentation, which consist layout drawing, drawing of truss for manufacturing, drawing indicative details and plan of anchorage, is a part od thesis

Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends

Flap endonucleases (FENs), essential for DNA replication and repair, recognize and remove RNA or DNA 5′-flaps. Related to FEN specificity for substrates with free 5′-ends, but controversial, is the role of the helical arch observed in varying conformations in substrate-free FEN structures. Conflicting models suggest either 5′-flaps thread through the arch, which when structured can only accommodate single-stranded (ss) DNA, or the arch acts as a clamp. Here we show that free 5′-termini are selected using a disorder-thread-order mechanism. Adding short duplexes to 5′-flaps or 3′-streptavidin does not markedly impair the FEN reaction. In contrast, reactions of 5′-streptavidin substrates are drastically slowed. However, when added to premixed FEN and 5′-biotinylated substrate, streptavidin is not inhibitory and complexes persist after challenge with unlabelled competitor substrate, regardless of flap length or the presence of a short duplex. Cross-linked flap duplexes that cannot thread through the structured arch react at modestly reduced rate, ruling out mechanisms involving resolution of secondary structure. Combined results explain how FEN avoids cutting template DNA between Okazaki fragments and link local FEN folding to catalysis and specificity: the arch is disordered when flaps are threaded to confer specificity for free 5′-ends, with subsequent ordering of the arch to catalyze hydrolysis

Mouse SLX4 Is a Tumor Suppressor that Stimulates the Activity of the Nuclease XPF-ERCC1 in DNA Crosslink Repair

SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents. Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks. Mini-SLX4-XPF-ERCC1 also vigorously stimulates dual incisions around a DNA crosslink embedded in a synthetic replication fork, an essential step in the repair of this lesion. These observations define vertebrate SLX4 as a tumor suppressor, which activates XPF-ERCC1 nuclease specificity in DNA crosslink repairope

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1 Catalyzed Reaction.

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5'-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5'-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5'-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5'-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Y40, D181 and R100 and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound, 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage

Sports hall

V rámci této bakalářské práce se budu zabývat návrhem nosné konstrukce sportovní haly pro běžné sporty (házená, malý fotbal, tenis, volejbal, košíková) situovanou v oblasti města Brno, přičemž půdorysné rozměry této konstrukce jsou cca 36 m x 48 m a světlé výšce cca 8 m. Příčnou vazbu haly tvoří vazníky, které jsou uloženy kloubově na čepovém spoji. Prostorové ztužení zajišťují příčná ztužidla. Součástí práce je výkresová dokumentace skládající se z dispozičního výkresu, příčných řezů, výrobního výkresu vazníku a vybraných detailů.This bachelor 's thesis deals with a project of roof load -bearing structure for common sports ( handball , football , tennis, volleyball , basketball ) of a sports hall situated in the city of Brno. Groun plan scantlings of the con - struction are of 36 x 48 m and clearance of 8 m. Hall travers bond is composed of trusses, which are hinged to pivot joints. Spatial rigidity is made safe due to sway bracings. The thesis includes documentation composed of layout drawing, cross section, drawing of production and drawing of selected details.

Orchestrating the nucleases involved in DNA interstrand cross-link (ICL) repair.

DNA interstrand cross-links (ICLs) pose a significant threat to genomic and cellular integrity by blocking essential cellular processes, including replication and transcription. In mammalian cells, much ICL repair occurs in association with DNA replication during S phase, following the stalling of a replication fork at the block caused by an ICL lesion. Here, we review recent work showing that the XPF-ERCC1 endonuclease and the hSNM1A exonuclease act in the same pathway, together with SLX4, to initiate ICL repair, with the MUS81-EME1 fork incision activity becoming important in the absence of the XPF-SNM1A-SLX4-dependent pathway. Another nuclease, the Fanconi anemia-associated nuclease (FAN1), has recently been implicated in the repair of ICLs, and we discuss the possible ways in which the activities of different nucleases at the ICL-stalled replication fork may be coordinated. In relation to this, we briefly speculate on the possible role of SLX4, which contains XPF and MUS81- interacting domains, in the coordination of ICL repair nucleases

The SNM1/Pso2 family of ICL repair nucleases: from yeast to man.

Efficient interstrand crosslink (ICL) repair in yeast depends on the Pso2/Snm1 protein. Pso2 is a member of the highly conserved metallo-beta-lactamase structural family of nucleases. Mammalian cells possess three SNM1/Pso2 related proteins, SNM1A, SNM1B/Apollo, and SNM1C/Artemis. Evidence that SNM1A and SNM1B contribute to ICL repair is mounting, whereas Artemis appears to primarily contribute to non-ICL repair pathways, particularly some double-strand break repair events. Yeast Pso2 and all three mammalian SNM1-family proteins have been shown to possess nuclease activity. Here, we review the biochemical, genetic, and cellular evidence for the SNM1 family as DNA repair factors, focusing on ICL repair

Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors