Podcasts about ssdna

Dan Wilson returns to TWiV to debunk vaccine misinformation by RFK Jr. during his recent appearance on the Joe Rogan Experience. Hosts: Vincent Racaniello, Dickson Despommier, Rich Condit, Kathy Spindler, and Brianne Barker Guest: Dan Wilson Subscribe (free): Apple Podcasts, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode MicrobeTV Discord Server Joe Rogan's worst misinformation yet, with RFK Jr. (YouTube) Every first vaccine has been tested in placebo-controlled trials before going to market. Polio Measles HPV COVID-19 HepB Haemophilus influenzae B Pertussis Diphtheria Tetanus Pneumococcal Mumps Meningococcal Chickenpox HepA KiGGS Study results on atopic diseases after vaccination (Vaccine) Vaccine history by year (CHOP) Financing vaccines in the 21st Century (Nat Acad Press) DALY rates from non-communicable diseases (Our World in Data) Science loses one to creationism (WaPo) Children's Health Defense (Wikipedia) COVID-19 vaccines saved 3 million US lives (CIDRAP) Beyond the Noise with Paul Offit (YouTube) Letters read on TWiV 1026 Timestamps by Jolene. Thanks! Weekly Picks Dickson – Jazz G.O.A.T.S – Louis Armstrong, Duke Ellington, Ella Fitzgerald Brianne – Defining the Anthropocene? Kathy – How to use your thermostat on A/C Rich – Star Trek: Strange New Worlds Vincent – Step Aside, Joe Biden Listener Picks Kim – Virus found in a boreal lake links ssDNA and dsDNA viruses and Structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv

Welcome to The Nonlinear Library, where we use Text-to-Speech software to convert the best writing from the Rationalist and EA communities into audio. This is: Biological DOOM: a brief overview of biological computation, published by Metacelsus on April 29, 2023 on LessWrong. (no, not that kind of biological doom) DOOM is a classic first-person shooter game released in 1993 by id Software. Because it's from 1993, it doesn't require much computing power compared to modern games. Additionally, the code (written in C) is easy to compile to run on a variety of processors. Over the years, hackers have made DOOM run on things such as an ATM, a touchbar of a MacBook, a Porsche 911, and even a TI-84 calculator powered by potato batteries. But what about cells? Requirements for DOOM The inputs to DOOM are based on button presses, traditionally on a keyboard. 9 keys in total are required (assuming “switch weapon” is implemented as one key that cycles through weapons). For computation, the original 1993 release required: 4 MB of RAM and 12 MB of hard-drive storage Intel 386 (bare minimum) or 486 processor. There is some flexibility regarding the processor, but slower processors will have worse frame-rates. The Intel 386 had 275,000 transistors in its most basic configuration. DOOM also requires a graphical output. The smallest resolution I've seen is 128x32 pixels, and that was cutting it a bit close. We'll assume we need 4096 black-and-white pixels for the display. Finally, DOOM has audio. For the purposes of this thought experiment, we can ignore this output. Although the soundtrack is great, it's not strictly required to play the game. Approaches to biological computation So, how could we potentially run DOOM? Biological systems can perform computations in several ways: Nucleic acid hybridization These logic gates are based on strand displacement between complementary DNA sequences. A recent paper demonstrated a set of DNA-based logic gates that could add two 6-bit binary numbers. Pros and cons: Memory capacity is good (encoded in DNA or RNA) Switching speed is OK (rate constants vary by design but are typically around 106M−1s−1) Visual output could be provided by fluorophore/quencher conjugated oligonucleotides, but . . . Coupling to a macroscopic output display would be far too slow, because it would have to rely on diffusion (taking a few minutes to cover a millimeter-scale distance). So, the game would have to be played using a microscope. It's hard to “reset” gates after using them, this requires coupling to some energy source It's also hard to integrate DNA-based logic gates into other biological systems, since not many organisms use short pieces of ssDNA. RNA might be used instead. Transcription and translation These logic gates use the same tools that cells use to regulate gene expression. For example, the classic lac operon in bacteria implements: Biologists have exploited similar systems to build logic gates, as well as systems involving the regulation of translation (the production of proteins using mRNAs as templates). A recent paper used Cas9 binding to a sgRNA-like sequence inserted in an mRNA to control its translation. To form a NAND gate, they split Cas9 into two fragments; if both were present, the output protein was not produced. Pros and cons: Memory capacity is acceptable (encoded in DNA or RNA) There will be challenges with implementing the number of logic gates required while avoiding cross-talk The dealbreaker: far too slow to run DOOM. RNA and protein half-lives are on the order of minutes to hours. Protein phosphorylation (kinases/phosphatases) Many cell signaling pathways use protein phosphorylation as a signal. This is much faster than transcription and translation, since no new RNAs or proteins need to be produced. A paper in 2021 built a toggle switch in yeast out of several kinases and phosphatases. Pros and cons: Response speed is adequate, similar to nucleic acid hybridization gates (i.e., largely l...

Link to original articleWelcome to The Nonlinear Library, where we use Text-to-Speech software to convert the best writing from the Rationalist and EA communities into audio. This is: Biological DOOM: a brief overview of biological computation, published by Metacelsus on April 29, 2023 on LessWrong. (no, not that kind of biological doom) DOOM is a classic first-person shooter game released in 1993 by id Software. Because it's from 1993, it doesn't require much computing power compared to modern games. Additionally, the code (written in C) is easy to compile to run on a variety of processors. Over the years, hackers have made DOOM run on things such as an ATM, a touchbar of a MacBook, a Porsche 911, and even a TI-84 calculator powered by potato batteries. But what about cells? Requirements for DOOM The inputs to DOOM are based on button presses, traditionally on a keyboard. 9 keys in total are required (assuming “switch weapon” is implemented as one key that cycles through weapons). For computation, the original 1993 release required: 4 MB of RAM and 12 MB of hard-drive storage Intel 386 (bare minimum) or 486 processor. There is some flexibility regarding the processor, but slower processors will have worse frame-rates. The Intel 386 had 275,000 transistors in its most basic configuration. DOOM also requires a graphical output. The smallest resolution I've seen is 128x32 pixels, and that was cutting it a bit close. We'll assume we need 4096 black-and-white pixels for the display. Finally, DOOM has audio. For the purposes of this thought experiment, we can ignore this output. Although the soundtrack is great, it's not strictly required to play the game. Approaches to biological computation So, how could we potentially run DOOM? Biological systems can perform computations in several ways: Nucleic acid hybridization These logic gates are based on strand displacement between complementary DNA sequences. A recent paper demonstrated a set of DNA-based logic gates that could add two 6-bit binary numbers. Pros and cons: Memory capacity is good (encoded in DNA or RNA) Switching speed is OK (rate constants vary by design but are typically around 106M−1s−1) Visual output could be provided by fluorophore/quencher conjugated oligonucleotides, but . . . Coupling to a macroscopic output display would be far too slow, because it would have to rely on diffusion (taking a few minutes to cover a millimeter-scale distance). So, the game would have to be played using a microscope. It's hard to “reset” gates after using them, this requires coupling to some energy source It's also hard to integrate DNA-based logic gates into other biological systems, since not many organisms use short pieces of ssDNA. RNA might be used instead. Transcription and translation These logic gates use the same tools that cells use to regulate gene expression. For example, the classic lac operon in bacteria implements: Biologists have exploited similar systems to build logic gates, as well as systems involving the regulation of translation (the production of proteins using mRNAs as templates). A recent paper used Cas9 binding to a sgRNA-like sequence inserted in an mRNA to control its translation. To form a NAND gate, they split Cas9 into two fragments; if both were present, the output protein was not produced. Pros and cons: Memory capacity is acceptable (encoded in DNA or RNA) There will be challenges with implementing the number of logic gates required while avoiding cross-talk The dealbreaker: far too slow to run DOOM. RNA and protein half-lives are on the order of minutes to hours. Protein phosphorylation (kinases/phosphatases) Many cell signaling pathways use protein phosphorylation as a signal. This is much faster than transcription and translation, since no new RNAs or proteins need to be produced. A paper in 2021 built a toggle switch in yeast out of several kinases and phosphatases. Pros and cons: Response speed is adequate, similar to nucleic acid hybridization gates (i.e., largely l...

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536536v1?rss=1 Authors: Di Biagi, L., Malacaria, E., Aiello, F. A., Valenzisi, P., Marozzi, G., Franchitto, A., Pichierri, P. Abstract: Replication gaps can arise as a consequence of perturbed DNA replication and their accumulation might undermine the stability of the genome. Loss of RAD52, a protein involved in the regulation of fork reversal, promotes accumulation of parental ssDNA gaps during replication perturbation. Here, we demonstrate that this is due to the engagement of Polalpha downstream of the extensive degradation of perturbed replication forks after their reversal, and is not dependent on PrimPol. Polalpha is hyper-recruited at parental ssDNA in the absence of RAD52, and this recruitment is dependent on fork reversal enzymes and RAD51. Of note, we report that the interaction between Polalpha and RAD51 is stimulated by RAD52 inhibition, and Polalpha-dependent gap accumulation requires nucleation of RAD51 suggesting that it occurs downstream strand invasion. Altogether, our data indicate that RAD51-Polalpha-dependent repriming is essential to promote fork restart and limit DNA damage accumulation when RAD52 function is disabled. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.04.367136v1?rss=1 Authors: Roy, U., Kwon, Y., Marie, L., Symington, L., Sung, P., Lisby, M., Greene, E. C. Abstract: Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA-bound states, the rate of which is dynamically controlled though the opposing actions of Rad55-Rad57 and Srs2. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.04.283879v1?rss=1 Authors: Ahmed, F., Patterson, A., Devershetty, J., Mattice, J., Pokhrel, N., Bothner, B., Antony, E. Abstract: Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes including DNA replication, repair, and recombination. RPA binds ssDNA utilizing six oligosaccharide/oligonucleotide binding (OB) domains within a heterotrimeric complex of RPA70, RPA32 and RPA14 subunits. Based on their DNA binding affinities they are classified as high versus low-affinity DNA binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS) we assessed the contacts and dynamics of the individual domains of human RPA to determine the landscape of conformational changes upon binding to ssDNA. As expected, ssDNA interacts with the major DBDs (A, B, C, and D). However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. DNA-dependent HDX kinetics are also captured for DBD-D and DBD-E. Slower allosteric changes transpire in the protein-protein interaction domains and the linker regions. We propose a dynamics-based DNA binding model for RPA utilizing a dynamic half and a less-dynamic half. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.278671v1?rss=1 Authors: Tompkins, K. J., Houtti, M., Litzau, L. A., Aird, E. J., Everett, B. A., Nelson, A. T., Pornschloegl, L., Limon-Swanson, L. K., Evans, R. L., Evans, K., Shi, K., Aihara, H., Gordon, W. R. Abstract: Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay termed HUH-seq to interrogate subtleties in Rep specificity, and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed us to engineer a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.26.269084v1?rss=1 Authors: El-Kamand, S., Jergic, S., Lawson, T., Kariawasam, R., Richard, D. J., Cubeddu, L., Gamsjaeger, R. Abstract: The oxidative modification of DNA can result in the loss of genome integrity and must be repaired to maintain overall genomic stability. We have recently demonstrated that human single stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays a crucial role in the removal of 8-oxo-7,8-dihydro-guanine (8-oxoG), the most common form of oxidative DNA damage. The ability of hSSB1 to form disulphide-bonded tetramers and higher oligomers in an oxidative environment is critical for this process. In this study, we have used nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR) experiments to determine the molecular details of ssDNA binding by oligomeric hSSB1. We reveal that hSSB1 oligomers can open up damaged dsDNA and interact with individual single strands; however, our data also show that oxidised bases are recognised in the same manner as undamaged DNA bases. NMR experiments provide evidence that oligomeric hSSB1 is able to bind longer ssDNA in both binding polarities using a distinct set of residues different to those of the related SSB from Escherichia coli. We further demonstrate that oligomeric hSSB1 recognises ssDNA with a significantly higher affinity than its monomeric form and propose structural models for oligomeric hSSB1-ssDNA interaction. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.11.247064v1?rss=1 Authors: Li, M., Sengupta, B., Benkovic, S. J., Lee, T. H., Hedglin, M. Abstract: Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the PCNA sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA damaging agents and is tightly regulated to restrict TLS to damaged DNA. RPA, the major single strand DNA (ssDNA) binding protein, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18-RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.214445v1?rss=1 Authors: Losakul, R., Tobar, D. E., Pimenov, A., Gutierrez, A., Schipper, R., Jehle, W. A., Postma, H. W. C. Abstract: Nanopores are an established paradigm in genome sequencing technology, with remarkable advances still being made today. All efforts continually address the challenges associated with rapid, accurate, high-throughput, and low cost detection, particularly with long-read length DNA. We report on the in situ melting and unzipping of long, high molecular weight DNA. At varying salt concentration, we directly compare the translocation conductance and speeds between SiN and graphene nanopores at sub-10 nm pore diameters. We observe the force-induced unzipping of dsDNA at higher salt concentrations than previously reported in literature. We observe free running translocation without secondary structures of ssDNA that is an order of magnitude longer than reported before. We hypothesize that the frayed single strands at the molecules end get captured with a higher likelihood than both ends together. In understanding this phenomena for long-read lengths, we continue to address the challenges revolving around future generations of sequencing technology. Copy rights belong to original authors. Visit the link for more info

BioTechniques Digital and Assistant Editors Abigail Sawyer and Tristan Free talk to Theo Roth from the University of California San Francisco Marson Lab (CA, USA). Having recently made headlines for his work in T-cell engineering, Theo takes us through his groundbreaking CRISPR research, its potential impacts in CAR-T cell therapy and further afield, whilst also tackling the evolving ethics in gene editing and the potential benefits of using ssDNA over dsDNA for CRISPR techniques.

The TWiV hosts present two potentially seminal papers, on long-distance chemoattraction of a host by a chlorovirus, and replication of a nanovirus across multiple cells in a plant. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Paul Has Measles now in German (virology blog) Chloroviruses lure hosts (J Virol) Multicellular replication in plants (eLife) Image credit Letters read on TWiV 539 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - Why Do So Many Scientists Want to be Filmmakers Alan- Better boarding method airlines won’t use Dickson- Mass timber building Kathy- What organisms to study flow chart Vincent - World Pulls Andon Cord on 737 MAX Listener Pick Richard- The Real Cost of Knowledge Islam- 200 icosahedral viruses poster Mike- Post-Doc Me Now Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiV hosts present two potentially seminal papers, on long-distance chemoattraction of a host by a chlorovirus, and replication of a nanovirus across multiple cells in a plant. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Paul Has Measles now in German (virology blog) Chloroviruses lure hosts (J Virol) Multicellular replication in plants (eLife) Image credit Letters read on TWiV 539 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - Why Do So Many Scientists Want to be Filmmakers Alan- Better boarding method airlines won’t use Dickson- Mass timber building Kathy- What organisms to study flow chart Vincent - World Pulls Andon Cord on 737 MAX Listener Pick Richard- The Real Cost of Knowledge Islam- 200 icosahedral viruses poster Mike- Post-Doc Me Now Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin The prolific podcast-shedding Hosts solve the case of the Global Health Intern with a snakelike lesion on her foot, and reveal the role of a single-stranded DNA binding protein in differentiation of trypanosomes. Become a patron of TWiP. Links for this episode: RPA protein and differentiation of T. cruzi (PLoS NTD) Image credit Letters read on TWiP 124 Case Study for TWiP 124 28 yo male from referral hospital near thai-burma border. Fever and chills 2 days, feels poorly, small amount of dark urine. Severe shaking chills, 1x per day, no rash. No diarrhea, difficulty breathing. Seen by local health care volunteer, went to hospital then tertiary hospital in Bangkok. Exposure history to pigs, dogs, insects, etc. Involved in timber industry and farming, sleeping out at night with no cover, clothes and sandals. No meds. Not married, family lives with him. Family is fine. Sleep in dwelling but no screens. No toxic habits, HIV negative, sexually active but not brothels. High fever, low bp, rapid heart rate, breathing rapidly, scleral icterus, dry mucus membranes, neck supple, lungs clear. 2/6 systolic murmur. Abdomen soft but tender, enlarged liver and spleen. Many cuts, bruises, bug bites. Labs: low platelets, low hematocrit, low glucose. Blood smear: abnormal, 5-10% infected RBCs with multiple band forms.  Send your case diagnosis, questions and comments to twip@microbe.tv

Fri, 30 Jan 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17985/ https://edoc.ub.uni-muenchen.de/17985/1/Witosch_Justine.pdf Witosch, Justine Martha ddc:540, ddc:500, Fakultät für

Posttranslational modifications (PTMs) of proteins by covalent attachment of functional groups (like phosphorylation, acetylation, methylation, glycosylation, etc.) are of key importance for the cell as they regulate various aspects of protein behavior after its synthesis, e.g., dictate protein interaction properties, change catalytic activity of enzymes, induce conformational changes, guide subcellular localization and determine protein stability. A special class of protein PTMs is the conjugation of small proteins of the ubiquitin family to typically acceptor lysine residues of the substrates. The reversible nature of this PTM and the presence of dedicated domains that specifically recognize modified substrates make this type of protein modification instrumental for the regulation of numerous biological pathways. For ubiquitylation, strong substrate selectivity due to the presence of highly diversified conjugation machinery is characteristic and well studied, especially in case of ubiquitin’s proteolytic role. On the contrary, much less is known about the principles of substrate specificity and mechanisms of PTM action in the ubiquitin-like protein SUMO modification system. Despite the fact that SUMOylation specifically targets hundreds of substrates and major conjugation steps are identical with ubiquitin system, strikingly only a handful of enzymes operate in the SUMO pathway, suggesting that other principles of substrate selectivity must apply and perhaps distinct mechanisms of PTM action exist in the SUMO pathway. Moreover, the recognition of SUMO modification is surprisingly simple and relies mainly on a short hydrophobic sequence known as SUMO-interacting motif (SIM), in striking contrast to the ubiquitin system, where numerous ubiquitin-binding domains exist with different interaction specificities. All these, together with the observations that SUMO conjugation machinery seems rather promiscuous in vitro, that typically only a small fraction of a protein is being SUMOylated at a given time, and that specific SUMOylation-defective mutants often exhibit no obvious phenotypes, whereas SUMO pathway mutants do, emphasize the question of substrate specificity in the SUMO system and suggest other principles of SUMO action on its substrates. Here, we address the question of SUMOylation specificity and function using DNA double-strand break (DSB) repair pathway via homologous recombination (HR) as a case study because of its strong ties to the SUMO system. First, using SILAC-based proteomic approach we show that proteins acting in the same DNA repair pathway become collectively SUMOylated upon a specific stimulus (HR factors – upon DSB induction; nucleotide excision repair factors – upon exposure to UV light), suggesting that SUMO machinery often targets protein groups within the same pathway. Then, focusing on the DSB repair we find that DNA-bound SUMO ligase Siz2 catalyzes collective multisite SUMOylation of a whole set of HR factors. Repair proteins are loaded onto resected single-stranded DNA (ssDNA) in the vicinity of the ligase, thus making exposure of ssDNA a precise trigger for modification. Protein group SUMOylation fosters physical interactions between the HR proteins engaged in DNA repair, because not only that they become collectively modified at multiple SUMO-acceptor sites, but they also possess multiple SIMs, which promote SUMO-SIM mediated complex formation. Only wholesale elimination of SUMOylation of the core HR proteins significantly affects the HR pathway by slowing down DNA repair, suggesting that SUMO acts synergistically on several proteins. Thus, we show that SUMOylation collectively targets functionally engaged protein group rather than individual proteins, whereas localization of modification enzymes and specific triggers ensure substrate specificity.

The integrity of the genome displays a central role for all living organisms. Double strand breaks (DSBs) are probably the most cytotoxic and hazardous type of DNA lesion and are linked to cancerogenic chromosome aberrations in humans. To maintain genome stability, cells use various repair mechanisms, including homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. The Mre11:Rad50 (MR) complex plays a crucial role in DSB repair processes including DSB sensing and processing but also tethering of DNA ends. The complex consists of the evolutionarily conserved core of two Rad50 ATPases from which a long coiled-coil region protrudes and a dimer of the Mre11 nuclease. Even though various enzymatic and also structural functions of MR(N) could be determined, so far the molecular interplay of Rad50´s ATPase together with DNA binding and processing by Mre11 is rather unclear. The crystal structure of the bacterial MR complex in its nucleotide free state revealed an elongated conformation with accessible Mre11 nuclease sites in the center and a Rad50 monomer on each outer tip, thus suggesting conformational changes upon ATP and/or DNA binding. However, so far high resolution structures of MR in its ATP and/or DNA bound state are lacking. The aim of this work was to understand the ATP-dependent engagement-disengagement cycle of Rad50´s nucleotide binding domains (NBDs) and thereby the ATP-controlled interaction between Mre11 and Rad50. For this purpose high resolution crystal structures of the bacterial Thermotoga maritima (Tm) MR complex with engaged Rad50 NBDs were determined. Small angle x-ray scattering proved the conformation of the nucleotide bound complex in solution. DNA affinity was also analyzed to investigate MR´s DNA binding mechanism. ATP binding to TmRad50 induces a large structural change and surprisingly, the NBD dimer binds directly in the Mre11 DNA binding cleft, thereby blocking Mre11’s dsDNA binding sites. DNA binding studies show that MR does not entrap DNA in a ring-like structure and that within the complex Rad50 likely forms a dsDNA binding site in response to ATP, while the Mre11 nuclease module retains ssDNA binding ability. Finally, a possible mechanism for ATP dependent DNA tethering and DSB processing by MR is proposed.

Dynamic remodeling of chromatin or other persistent protein:DNA complexes is essential for genome expression and maintenance. Proteins of the SWI2/SNF2 family catalyze rearrangements of diverse protein:DNA complexes. Although SWI2/SNF2 enzymes exhibit a diverse domain organisation, they share a conserved catalytic ATPase domain that is related to superfamily II helicases through the presence of seven conserved sequence motifs. In contrast to helicases, SWI2/SNF2 enzymes lack helicase activity, but use ATP hydrolysis to translocate on DNA and to generate superhelical torsion into DNA. How these features implicate remodeling function or how ATP hydrolysis is coupled to these rearrangements is poorly understood and suffers from the lack of structural information regarding the catalytic domain of SWI2/SNF2 ATPase In this PhD thesis I characterized the catalytic domain of Sulfolobus solfataricus Rad54 homolog (SsoRad54cd). Like the eukaryotic SWI2/SNF2 ATPases, SsoRad54cd exhibits dsDNA stimulated ATPase activity, lacks helicase activity and has dsDNA translocation and distortion activity. These activities are thereby features of the conserved catalytic ATPase domain itself. Furthermore, the crystal structures of SsoRad54cd in absence and in complex with its dsDNA substrate were determined. The Sulfolobus solfataricus Rad54 homolog catalytic domain consists of two RecA-like domains with two helical SWI2/SNF2 specific subdomains, one inserted in each domain. A deep cleft separates the two domains. Fully base paired duplex DNA binds along the domain 1: domain 2 interface in a position, where rearrangements of the two RecA-like domains can directly be translated in DNA manipulation. The binding mode of DNA to SsoRad54cd is consistent with an enzyme that translocate along the minor groove of DNA. The structure revealed a remarkable similarity to superfamily II helicases. The related composite ATPase active site as well as the mode of DNA recognition suggests that ATP-driven transport of dsDNA in the active site of SWI2/SNF2 enzymes is mechanistically related to ATP-driven ssDNA in the active site of helicases. Based on structure-function analysis a specific model for SWI2/SNF2 function is suggested that links ATP hydrolysis to dsDNA translocation and DNA distortion. The represented results have structural implications for the core mechanism of remodeling factors. If SWI2/SNF2 ATPases are anchored to the substrate protein:DNA complex by additional substrate interacting domains or subunits, ATP-driven cycles of translocation could transport DNA towards or away from the substrate or generate torsional stress at the substrate:DNA interface. Finally, I provide a molecular framework for understanding mutations in Cockayne and X-linked mental retardation syndromes. Mapping of the mutations on the structure of SsoRad54cd reveal that the mutations colocalize in two surface clusters: Cluster I is located adjacent to a hydrophobic surface patch that may provide a macromolecular interaction site. Cluster II is situated in the domain 1 : domain 2 interface near the proposed pivot region and may interfere with ATP driven conformational changes between domain 1 and domain 2.

The product of the ie 1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS–PAGe, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as -galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions.