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Structural and functional studies of the restriction endonuclease BpuJI / Restrikcijos endonukleazės BpuJI struktūriniai ir ir funkciniai tyrimaiSukackaitė, Rasa 15 December 2009 (has links)
Type II restriction endonucleases recognize specific DNA sequences and cleave DNA at fixed positions within or close to this sequence. BpuJI recognizes the 5’-CCCGT sequence, but in contrast to other enzymes its cleavage site is very variable. This study shows that BpuJI is a dimer in solution and consists of two separate domains. The N-domain binds to the target sequence as a monomer, while the C-domain is responsible for nuclease activity and dimerization. The nuclease activity is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the N-domains. The activated C-domain cleaves DNA near the target site. In addition, it possesses an end-directed nuclease activity and preferentially cuts ~3 nt from the 3’ terminus. This leads to a very complicated pattern of DNA cleavage. Bioinformatics and mutational analysis revealed that the BpuJI C-domain harbours a PD (D/E)XK active site and is structurally related to archaeal Holliday junction resolvases. The crystal structure of the BpuJI N-domain bound to cognate DNA was solved at 1.3 Å resolution. It revealed two winged-helix subdomains, D1 and D2. The recognition of the target sequence is achieved the amino acid residues located on both the HTH motifs and an N-terminal arm. The BpuJI DNA recognition domain is most similar to the nicking endonuclease Nt.BspD6I. The modelling suggests that Nt.BspD6I could share the specificity-determining regions with BpuJI. / II tipo restrikcijos endonukleazės atpažįsta specifines DNR sekas ir kerpa DNR šiose sekose arba šalia jų. BpuJI, atpažįstanti 5’-CCCGT seką, skiriasi nuo kitų fermentų tuo, kad jos kirpimo vieta yra labai variabili. Čia parodoma, kad BpuJI yra dimeras, sudarytas iš dviejų monomerų, kurie turi po du atskirus domenus. BpuJI N domenas atpažįsta taikinį kaip monomeras, o C-domenas pasižymi nukleaziniu aktyvumu ir dimerizuojasi. Apo-fermento nukleazinis aktyvumas yra nuslopintas. N-domenams atpažinus taikinį, aktyvuojamas C-domenas, kuris perkerpa DNR šalia taikinio. Be to, aktyvuotas C-domenas yra nespecifinė nukleazė, linkusi nukirpti ~3 nt nuo buko dvigrandės DNR galo. Taigi, BpuJI DNR karpymo pobūdis yra labai sudėtingas. Bioinformatinė analizė ir kryptinga mutagenezė parodė, kad BpuJI C-domenas turi PD-(D/E)XK struktūrinę sanklodą ir yra panašus į archėjų Holidėjaus jungtis karpančias nukleazes. Išsprendus 1,3 Å skiriamosios gebos BpuJI N-domeno/DNR komplekso erdvinė struktūrą, paaiškėjo, kad šį domeną sudaro du „sparnuotą“ spiralė-linkis-spiralė motyvą turintys subdomenai. BpuJI taikinį atpažįsta aminorūgštys, esančios N-rankoje ir abiejų spiralė-linkis-spiralė motyvų atpažinimo spiralėse. BpuJI N-domenas yra labiausiai panašus į Nt.BspD6I nukleazę, kerpančią vieną DNR grandinę. Nt.BspD6I/DNR komplekso struktūros modelis rodo, kad Nt.BspD6I ir BpuJI taikinį atpažįstantys struktūriniai elementai yra panašūs.
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Molecular Dynamics Studies of the Phi29 Connector-DNA complexKumar, Rajendra 18 July 2014 (has links)
No description available.
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A Structural and Mechanistic Study of Two Members of Cupin Family ProteinLiu, Fange 18 June 2013 (has links)
is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses.
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Large scale simulations of genome organisation in living cellsJohnson, James January 2018 (has links)
Within every human cell, approximately two meters of DNA must be compacted into a nucleus with a diameter of around ten micrometers. Alongside this daunting storage problem, the 3D organisation of the genome also helps determine which genes are up- or down-regulated, which in turn effects the functionality of the cell itself. While the organisational structure of the genome can be revealed using experimental techniques such as chromosome conformation capture and its high-throughput variant Hi-C, the mechanisms driving this organisation are still unclear. The first two results chapters of this thesis use molecular dynamics simulations to investigate the effect of a potential organisational mechanisms for DNA known as the "bridging-induced attraction". This mechanism involves multivalent DNA-binding proteins bridging genomically distant regions of DNA, which in turn promotes further binding of proteins and compaction of the DNA. In chapter 2 (the first results chapter) we look at a model where proteins can bind non-specifically to DNA, leading to cluster formation for suitable protein-DNA interaction strengths. We also show the effects of protein concentration on the DNA, with a collapse from a swollen to a globular phase observed for suitably high protein concentrations. Chapter 3 develops this model further, using genomic data from the ENCODE project to simulate the "specific binding" of proteins to either active (euchromatin) or inactive (heterochromatin) regions. We were then able to compare contact maps for specific simulated chromosomes with the experimental Hi-C data, with our model reproducing well the topologically associated domains (TADs) seen in Hi-C contact maps. In chapter 4 of the thesis we use numerical methods to study a model for the coupling between DNA topology (in particular, supercoiling in DNA and chromatin) and transcription in a genome. We present details of this model, where supercoiling flux is induced by gene transcription, and can diffuse along the DNA. The probability of transcription is also related to supercoiling, as regions of DNA which are negatively supercoiled have a greater likelihood of being transcribed. By changing the magnitude of supercoiling flux, we see a transition between a regime where transcription is random and a regime where transcription is highly correlated. We also find that divergent gene pairs show increased transcriptional activity, along with transcriptional waves and bursts in the highly correlated regime { all these features are associated with genomes of living organisms.
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DNA Binding Studies With The Transcriptional Activator Protein C Of Bacteriophage MURamesh, V 10 1900 (has links) (PDF)
No description available.
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A Single Molecule Perspective on Protein-DNA CondensatesRenger, Roman 22 December 2020 (has links)
Biomolecular condensates are dynamic intracellular structural units or distinct reaction spaces that can form by condensation of their constituents from the cytoplasm or the nucleoplasm. It is generally not clear yet, how dynamic, continuum-like condensate properties relevant for large-scale intracellular organisation emerge from the interplay of proteins and nucleic acids on the level of few individual molecules. With this work, we expand the portfolio of methods to investigate the role of protein-nucleic acid interactions in biomolecular condensates by introducing optical tweezers-based mechanical micromanipulation of single DNA molecules combined with confocal fluorescence microscopy to the field. We used this approach to characterise how the two landmark proteins1 Fused in Sarcoma and Heterochromatin Protein 1 form condensates with single DNA molecules. Fused in Sarcoma (FUS) is a key protein for various aspects of the nucleic acid metabolism and evidence is accumulating that biomolecular condensation is crucial for both, its physiological functions and its role in pathological aggregate formation. In this thesis, we directly visualised the formation of FUS condensates with single molecules of ssDNA and dsDNA. We showed that the formation of these microcondensates is based on nucleic acid scaffolding. We explored their mechanical properties and found that the mechanical tension that (FUS dsDNA) condensates can withstand or exert is in the range below 2 pN. We further demonstrated that already on this fundamental scale and with limited amounts of constituent molecules, dynamic properties like shape relaxations, reminiscent of viscoelastic materials, can emerge. Heterochromatin Protein 1 (HP1) is a prototypic chromatin organising factor that is in particular involved in the formation of dynamically compacted heterochromatin domains. HP1 forms biomolecular condensates and compacts DNA strands in vitro. In this work, we measured the influence of HP1 on the
mechanical properties of individual DNA molecules and demonstrated the response of HP1-DNA condensates to different environmental conditions. We contributed a methodological framework to characterise viscoelastic-like systems on the single molecule level.
Taken together, our optical tweezers-based approach revealed structural and mechanical properties of prototypic protein-DNA condensates and hence helped to elucidate mechanisms underlying their formation in unprecedented spatiotemporal and mechanical detail. We anticipate that this method can become a valuable tool to investigate how large-scale intracellular organisation based on protein-nucleic acid condensation emerges from interactions between individual building blocks.
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Characterization of Prokaryotic Ku DNA Binding PropertiesKoechlin, Lucas January 2020 (has links)
DNA damage occurs to all living things; its subsequent repair is a crucial component of life. The most dangerous, and potentially most useful form of DNA damage is the double strand break (DSB). A DSB is defined by breaks occurring to both sugar phosphate backbones in close enough proximity that they lead to the separation of the two pieces of the DNA. This type of damage will kill the cell if left unrepaired. It is the most lethal type of DNA damage.
Most living organisms have also developed ways to take advantage of DSBs through their repair systems, primarily as a means of introducing genetic variation. There are two primary DSB repair pathways across life: homologous recombination (HR) and non-homologous end-joining (NHEJ). The focus of this work is NHEJ. NHEJ is known as “error-prone” because it does not use a homologous template and can introduce small addition or deletion mutations during the repair process. This pathway has been extensively studied in eukaryotes and is known as the primary form of DSB repair in mammalian cells, however the prokaryotic NHEJ system was more recently identified and as a result, a void of information surrounds it.
NHEJ is comprised of 3 core steps: DSB recognition and binding, DNA end processing, and ligation. In the eukaryotic version of NHEJ these 3 steps involve a plethora of factors; conversely, in the prokaryotic version, the same functionality is accomplished by just 2 proteins, bacterial Ku and LigD. The focus of this research is Ku: the DNA end-binding protein responsible for identifying the DSB, binding and protecting the DNA end, as well as recruiting LigD to the break. Ku is composed of 2 domains, the first of which is predicted to be highly homologous to eukaryotic Ku’s equivalent domain; this is the core domain which forms a ring-like structure that DNA threads through. The second is completely unique to bacterial Ku, it is the C-terminal domain, which can further be split into 2 sub-domains, the minimal C-terminus, and the extended C-terminus. The sub-domains are defined by their level of conservation across bacterial species, with the minimal C-terminus being highly conserved, while the extended C-terminus is highly variable. Using DNA-binding assays and several mutant constructs which affect the C-terminal domain, I show that this C-terminus is unexpectedly responsible for destabilizing the Ku-DNA interaction. This observation leads me to hypothesize that maintaining a weak interaction with DNA is important for Ku because of the other proteins which need access to the DNA (e.g. replicative helicase). While Ku is bound, it could be capable of blocking regions of DNA, in turn blocking other vital cellular processes like replication. Ku maintaining a lower affinity for DNA should facilitate Ku displacement by other proteins. A tighter binding would restrict Ku’s freedom to move on DNA making it more likely to inhibit other critical pathways. To better understand Ku, I attempted to solve the Ku structure using X-ray crystallography, and was able to achieve crystals of Ku, however diffraction was too limited for a structure. Another way to investigate the validity of my proposed model is to use a biophysical approach with atomic force microscopy (AFM) to visualize protein-DNA complexes. The initial work has established key controls for future Ku-DNA AFM work by imaging and analyzing Ku on its own. Interest in bacterial NHEJ is two-fold from the antimicrobial perspective: NHEJ is a highly mutagenic pathway, so it serves as a proverbial well for differentiation and thus the development of antimicrobial resistance (AMR); NHEJ is very important in bacteria that enter a stationary phase due to their lack of a homologous piece of DNA for HR. Thus, NHEJ inhibition could be useful for slowing bacterial evolution and potentially as a treatment for infections such a Mycobacterium tuberculosis, which is known to lie dormant in host macrophages for long periods of time. To investigate the viability of NHEJ inhibition, I had begun the process of creating ∆ku strains of Pseudomonas aeruginosa to simulate Ku inhibition under various conditions. This Ku project is the focus of the first two chapters, however, during my Master’s degree I participated in 2 other major projects. The third chapter details a bacterial DNA damage tolerance pathway, which similarly is highly mutagenic and poorly characterized: the ImuABC translesion synthesis polymerase complex. The fourth and final chapter details the work for a Journal of Visualized Experiments article meant to highlight the benefits of AFM as a means of studying protein-DNA interactions. / Thesis / Master of Science (MSc)
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Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpaPetzold, Herman E. III 09 November 2017 (has links)
The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield. / Ph. D.
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From network to pathway: integrative network analysis of genomic dataWang, Chen 25 August 2011 (has links)
The advent of various types of high-throughput genomic data has enabled researchers to investigate complex biological systems in a systemic way and started to shed light on the underlying molecular mechanisms in cancers. To analyze huge amounts of genomic data, effective statistical and machine learning tools are clearly needed; more importantly, integrative approaches are especially needed to combine different types of genomic data for a network or pathway view of biological systems. Motivated by such needs, we make efforts in this dissertation to develop integrative framework for pathway analysis. Specifically, we dissect the molecular pathway into two parts: protein-DNA interaction network and protein-protein interaction network. Several novel approaches are proposed to integrate gene expression data with various forms of biological knowledge, such as protein-DNA interaction and protein-protein interaction for reliable molecular network identification.
The first part of this dissertation seeks to infer condition-specific transcriptional regulatory network by integrating gene expression data and protein-DNA binding information. Protein-DNA binding information provides initial relationships between transcription factors (TFs) and their target genes, and this information is essential to derive biologically meaningful integrative algorithms. Based on the availability of this information, we discuss the inference task based on two different situations:
(a) if protein-DNA binding information of multiple TFs is available:
based on the protein-DNA data of multiple TFs, which are derived from sequence analysis between DNA motifs and gene promoter regions, we can construct initial connection matrix and solve the network inference using a constraint least-squares approach named motif-guided network component analysis (mNCA). However, connection matrix usually contains a considerable amount of false positives and false negatives that make inference results questionable. To circumvent this problem, we propose a knowledge based stability analysis (kSA) approach to test the conditional relevance of individual TFs, by checking the discrepancy of multiple estimations of transcription factor activity with respect to different perturbations on the connections. The rationale behind stability analysis is that the consistency of observed gene expression and true network connection shall remain stable after small perturbations are applied to initial connection matrix. With condition-specific TFs prioritized by kSA, we further propose to use multivariate regression to highlight condition-specific target genes. Through simulation studies comparing with several competing methods, we show that the proposed schemes are more sensitive to detect relevant TFs and target genes for network inference purpose. Experimentally, we have applied stability analysis to yeast cell cycle experiment and further to a series of anti-estrogen breast cancer studies. In both experiments not only biologically relevant regulators are highlighted, the condition-specific transcriptional regulatory networks are also constructed, which could provide further insights into the corresponding cellular mechanisms.
(b) if only single TF's protein-DNA information is available:
this happens when protein-DNA binding relationship of individual TF is measured through experiments. Since original mNCA requires a complete connection matrix to perform estimation, an incomplete knowledge of single TF is not applicable for such approach. Moreover, binding information derived from experiments could still be inconsistent with gene expression levels. To overcome these limitations, we propose a linear extraction scheme called regulatory component analysis (RCA), which can infer underlying regulation relationships, even with partial biological knowledge. Numerical simulations show significant improvement of RCA over other traditional methods to identify target genes, not only in low signal-to-noise-ratio situations and but also when the given biological knowledge is incomplete and inconsistent to data. Furthermore, biological experiments on Escherichia coli regulatory network inferences are performed to fairly compare traditional methods, where the effectiveness and superior performance of RCA are confirmed.
The second part of the dissertation moves from protein-DNA interaction network up to protein-protein interaction network, to identify dys-regulated protein sub-networks by integrating gene expression data and protein-protein interaction information. Specifically, we propose a statistically principled method, namely Metropolis random walk on graph (MRWOG), to highlight condition-specific PPI sub-networks in a probabilistic way. The method is based on the Markov chain Monte Carlo (MCMC) theory to generate a series of samples that will eventually converge to some desired equilibrium distribution, and each sample indicates the selection of one particular sub-network during the process of Metropolis random walk. The central idea of MRWOG is built upon that the essentiality of one gene to be included in a sub-network depends on not only its expression but also its topological importance. Contrasted to most existing methods constructing sub-networks in a deterministic way and therefore lacking relevance score for each protein, MRWOG is capable of assessing the importance of each individual protein node in a global way, not only reflecting its individual association with clinical outcome but also indicating its topological role (hub, bridge) to connect other important proteins. Moreover, each protein node is associated with a sampling frequency score, which enables the statistical justification of each individual node and flexible scaling of sub-network results. Based on MRWOG approach, we further propose two strategies: one is bootstrapping used for assessing statistical confidence of detected sub-networks; the other is graphic division to separate a large sub-network to several smaller sub-networks for facilitating interpretations. MRWOG is easy to use with only two parameters need to be adjusted, one is beta value for performing random walk and another is Quantile level for calculating truncated posteriori mean. Through extensive simulations, we show that the proposed scheme is not sensitive to these two parameters in a relatively wide range. We also compare MRWOG with deterministic approaches for identifying sub-network and prioritizing topologically important proteins, in both cases MRWG outperforms existing methods in terms of both precision and recall. By utilizing MRWOG generated node/edge sampling frequency, which is actually posteriori mean of corresponding protein node/interaction edge, we illustrate that condition-specific nodes/interactions can be better prioritized than the schemes based on scores of individual node/interaction. Experimentally, we have applied MRWOG to study yeast knockout experiment for galactose utilization pathways to reveal important components of corresponding biological functions; we also applied MRWSOG to study breast cancer patient prognostics problems, where the sub-network analysis could lead to an understanding of the molecular mechanisms of antiestrogen resistance in breast cancer.
Finally, we conclude this dissertation with a summary of the original contributions, and the future work for deepening the theoretical justification of the proposed methods and broadening their potential biological applications such as cancer studies. / Ph. D.
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Die Restriktionsendonuklease EcoRII: Primitives antivirales Abwehrsystem der Bakterien oder mehr?Reuter, Monika 20 November 2002 (has links)
Bakterielle Restriktions- und Modifikationssysteme (R/M-Systeme) greifen DNA endonukleolytisch an, die nicht die spezifische Markierung der eigenen Wirtszelle trägt. Zu einem R/M-System gehören eine Restriktionsendonuklease und eine DNA- Methyltransferase gleicher DNA-Spezifität. Die biologische Funktion der Restriktionsendonuklease besteht in der Abwehr von fremder, in die Zelle eindringender DNA, z. B? von Virus-Infektionen. Die korrespondierende DNA-Methyltransferase schützt die zelluläre DNA durch sequenz-spezifische DNA-Methylierung vor der endonukleolytischen Wirkung der Restriktionsendonuklease. Die dimeren TypII- Restriktionsendonukleasen erkennen kurze spezifische, unmethylierte Basensequenzen, die sie in Anwesenheit von Mg2+ Ionen an einer definierten Position endonukleolytisch spalten. Die Restriktionsendonuklease EcoRII braucht die koordinierte Wechselwirkung mit zwei Kopien der Sequenz 5 CCWGG, um katalytisch aktiv sein zu können, wobei eine der beiden Sequenzen als allosterischer Effektor wirkt und nicht gespalten werden muß. Die zwei Kopien der 5 CCWGG Sequenz können sowohl auf demselben als auch auf verschiedenen Molekülen lokalisiert sein. Die Interaktion von EcoRII mit verschiedenen DNA-Molekülen ist durch deren Länge und Konzentration, die Interaktion innerhalb eines DNA-Moleküls durch den Abstand zwischen beiden Sequenzen limitiert. Die durch Proteolyse nachgewiesene Zwei-Domänen-Struktur von EcoRII scheint diese besondere Form der Protein-DNA-Wechselwirkung zu ermöglichen. Die C-terminale Domäne von EcoRII stellt eine neue Restriktionsendonuklease (EcoRII-C) dar. Im Gegensatz zum Wildtyp-Enzym spaltet EcoRII-C an singulären 5 CCWGG Sequenzen. Die trunkierte Endonuklease spaltet DNA spezifisch und unabhängig von einem zweiten EcoRII-Erkennungsort. Die Reaktion verläuft deutlich schneller als die des kompletten EcoRII-Proteins. Die N-terminale Domäne bindet spezifisch DNA, attenuiert die endonukleolytische Aktivität von EcoRII und macht das Enzym abhängig von einer zweiten Kopie der Sequenz 5 CCWGG. EcoRII Wildtyp könnte demzufolge ein evolutionäres Intermediat zwischen einer sequenz-spezifischen Endonuklease und einem Protein sein, das spezifisch mit zwei Orten auf der DNA interagiert, wie z. B. Rekombinasen oder Transposasen. Durch die Kombination beider Funktionen könnte EcoRII selbst die Verbreitung der EcoRII-codierenden DNA-Sequenz in neue Populationen, ähnlich einem transponiblen Element, realisieren. / Bacterial restriction and modification systems (R/M-systems) endonucleolytically attack DNA that is not host cell-specifically modified. R/M-systems comprise a restriction endonuclease and a DNA methyltransferase exhibiting the same DNA sequence specificity. The biological function of the restriction endonuclease is the protection of the cell against invading foreign DNA, e. g. virus infection. The corresponding DNA methyltransferase renders cellular DNA resistent against the endonucleolytic action of the restriction endonuclease by sequence-specific DNA methylation. Dimeric type II- restriction endonucleases recognize short, specific, and unmethylated base sequences that they cut at a defined position in the presence of Mg2+ ions. Restriction endonuclease EcoRII requires the co- ordinated interaction with two copies of the sequence 5 CCWGG for catalytic activity. One of these sequences serves as an allosteric activator site and has not to be cleaved. The two copies of the sequence 5 CCWGG can be located as well on the same as on different DNA molecule(s). EcoRII interaction with two sites on different DNA molecules is limited by their length and concentration, EcoRII interaction within one DNA molecule is limited by the distance between the two sites. The two- domain structure of EcoRII figured out by limited proteolysis studies probably allows this particular form of protein-DNA interaction. The C-terminal domain of EcoRII represents a new restriction endonuclease (EcoRII-C). In contrast to EcoRII wild type, EcoRII-C cleaves DNA at single 5 CCWGG sites. The truncated endonuclease cleaves DNA specifically and independent of a second site. The enzymatic reaction passes well more rapid than that of the complete enzyme. The N-terminal domain binds DNA specifically, attenuates the endonucleolytic activity of EcoRII and makes it dependent on a second copy of the sequence 5 CCWGG. Therefore, the current EcoRII could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites on the DNA such as recombinases and transposases. The combination of both functions may enable EcoRII to accomplish its own propagation similarly to transposable elements.
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