Dokument-ID Dokumenttyp Verfasser/Autoren Herausgeber Haupttitel Abstract Auflage Verlagsort Verlag Erscheinungsjahr Seitenzahl Schriftenreihe Titel Schriftenreihe Bandzahl ISBN Quelle der Hochschulschrift Konferenzname Quelle:Titel Quelle:Jahrgang Quelle:Heftnummer Quelle:Erste Seite Quelle:Letzte Seite URN DOI Abteilungen OPUS4-19121 Wissenschaftlicher Artikel Daryaee, Fereidoon; Chang, Andrew; Schiebel, Johannes; Lu, Yang; Zhang, Zhuo; Kapilashrami, Kanishk; Walker, Stephen G.; Kisker, Caroline; Sotriffer, Christoph A.; Fisher, Stewart L.; Tonge, Peter J. Correlating drug-target kinetics and in vivo pharmacodynamics: long residence time inhibitors of the FabI enoyl-ACP reductase Drug-target kinetics enable time-dependent changes in target engagement to be quantified as a function of drug concentration. When coupled to drug pharmacokinetics (PK), drug-target kinetics can thus be used to predict in vivo pharmacodynamics (PD). Previously we described a mechanistic PK/PD model that successfully predicted the antibacterial activity of an LpxC inhibitor in a model of Pseudomonas aeruginosa infection. In the present work we demonstrate that the same approach can be used to predict the in vivo activity of an enoyl-ACP reductase (FabI) inhibitor in a model of methicillin-resistant Staphylococcus aureus (MRSA) infection. This is significant because the LpxC inhibitors are cidal, whereas the FabI inhibitors are static. In addition P. aeruginosa is a Gram-negative organism whereas MRSA is Gram-positive. Thus this study supports the general applicability of our modeling approach across antibacterial space. 2016 5945-5954 Chemical Science 7 9 urn:nbn:de:bvb:20-opus-191218 10.1039/c6sc01000h Rudolf-Virchow-Zentrum OPUS4-17540 Wissenschaftlicher Artikel Rohleder, Florian; Huang, Jing; Xue, Yutong; Kuper, Jochen; Round, Adam; Seidman, Michael; Wang, Weidong; Kisker, Caroline FANCM interacts with PCNA to promote replication traverse of DNA interstrand crosslinks FANCM is a highly conserved DNA remodeling enzyme that promotes the activation of the Fanconi anemia DNA repair pathway and facilitates replication traverse of DNA interstrand crosslinks. However, how FANCM interacts with the replication machinery to promote traverse remains unclear. Here, we show that FANCM and its archaeal homolog Hef from Thermoplasma acidophilum interact with proliferating cell nuclear antigen (PCNA), an essential co-factor for DNA polymerases in both replication and repair. The interaction is mediated through a conserved PIP-box; and in human FANCM, it is strongly stimulated by replication stress. A FANCM variant carrying a mutation in the PIP-box is defective in promoting replication traverse of interstrand crosslinks and is also inefficient in promoting FANCD2 monoubiquitination, a key step of the Fanconi anemia pathway. Our data reveal a conserved interaction mode between FANCM and PCNA during replication stress, and suggest that this interaction is essential for FANCM to aid replication machines to traverse DNA interstrand crosslinks prior to post-replication repair. 2016 3219–3232 Nucleic Acids Research 44 7 urn:nbn:de:bvb:20-opus-175401 10.1093/nar/gkw037 Rudolf-Virchow-Zentrum OPUS4-11947 Wissenschaftlicher Artikel Schmitt, Dominik R.; Kuper, Jochen; Elias, Agnes; Kisker, Caroline The Structure of the TFIIH p34 Subunit Reveals a Von Willebrand Factor A Like Fold RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH). A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA) like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested. 2014 e102389 PLoS ONE 9 7 urn:nbn:de:bvb:20-opus-119471 10.1371/journal.pone.0102389 Julius-von-Sachs-Institut für Biowissenschaften OPUS4-11463 Wissenschaftlicher Artikel Khan, Irfan; Suhasini, Avvaru N.; Banerjee, Taraswi; Sommers, Joshua A.; Kaplan, Daniel L.; Kuper, Jochen; Kisker, Caroline; Brosh, Jr., Robert M. Impact of Age-Associated Cyclopurine Lesions on DNA Repair Helicases 8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair. 2014 e113293 PLOS ONE 9 11 urn:nbn:de:bvb:20-opus-114635 10.1371/journal.pone.0113293 Rudolf-Virchow-Zentrum OPUS4-31182 unpublished Neitz, Hermann; Bessi, Irene; Kuper, Jochen; Kisker, Caroline; Höbartner, Claudia Programmable DNA interstrand crosslinking by alkene-alkyne [2+2] photocycloaddition Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2'-deoxyuridine (\(^{Phe}\)dU). The crosslinking ability of \(^{Phe}\)dU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2+2] cycloaddition within the DNA duplex, resulting in efficient formation of a \(^{Phe}\)dU-dimer after short irradiation times of a few seconds. A \(^{Phe}\)dU dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology. submitted version 2023 Journal of the American Chemical Society urn:nbn:de:bvb:20-opus-311822 10.1021/jacs.3c01611 Rudolf-Virchow-Zentrum OPUS4-17017 Wissenschaftlicher Artikel Radu, Laura; Schoenwetter, Elisabeth; Braun, Cathy; Marcoux, Julien; Koelmel, Wolfgang; Schmitt, Dominik R.; Kuper, Jochen; Cianférani, Sarah; Egly, Jean M.; Poterszman, Arnaud; Kisker, Caroline The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy. 2017 10872-10883 Nucleic Acids Research 45 18 urn:nbn:de:bvb:20-opus-170173 10.1093/nar/gkx743 Rudolf-Virchow-Zentrum OPUS4-17076 Wissenschaftlicher Artikel Kaiser, Sebastian; Sauer, Florian; Kisker, Caroline The structural and functional characterization of human RecQ4 reveals insights into its helicase mechanism RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members. 2017 Nature Communications 8 15907 urn:nbn:de:bvb:20-opus-170769 10.1038/ncomms15907 Rudolf-Virchow-Zentrum OPUS4-23180 Wissenschaftlicher Artikel Petruseva, Irina; Naumenko, Natalia; Kuper, Jochen; Anarbaev, Rashid; Kappenberger, Jeannette; Kisker, Caroline; Lavrik, Olga The Interaction Efficiency of XPD-p44 With Bulky DNA Damages Depends on the Structure of the Damage The successful elimination of bulky DNA damages via the nucleotide excision repair (NER) system is largely determined by the damage recognition step. This step consists of primary recognition and verification of the damage. The TFIIH helicase XPD plays a key role in the verification step during NER. To date, the mechanism of damage verification is not sufficiently understood and requires further detailed research. This study is a systematic investigation of the interaction of ctXPD (Chaetomium thermophilum) as well as ctXPD-ctp44 with model DNAs, which contain structurally different bulky lesions with previously estimated NER repair efficiencies. We have used ATPase and DNA binding studies to assess the interaction of ctXPD with damaged DNA. The result of the analysis of ctXPD-ctp44 binding to DNA containing fluorescent and photoactivatable lesions demonstrates the relationship between the affinity of XPD for DNAs containing bulky damages and the ability of the NER system to eliminate the damage. Photo-cross-linking of ctXPD with DNA probes containing repairable and unrepairable photoactivatable damages reveals differences in the DNA interaction efficiency in the presence and absence of ctp44. In general, the results obtained indicate the ability of ctXPD-ctp44 to interact with a damage and suggest a significant role for ctp44 subunit in the verification process. 2021 Frontiers in Cell and Developmental Biology 9 urn:nbn:de:bvb:20-opus-231806 10.3389/fcell.2021.617160 Rudolf-Virchow-Zentrum OPUS4-22985 Wissenschaftlicher Artikel Peissert, Stefan; Sauer, Florian; Grabarczyk, Daniel B.; Braun, Cathy; Sander, Gudrun; Poterszman, Arnaud; Egly, Jean-Marc; Kuper, Jochen; Kisker, Caroline In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold. 2020 Nature Communications 11 1 urn:nbn:de:bvb:20-opus-229857 10.1038/s41467-020-15241-9 Medizinische Klinik und Poliklinik II OPUS4-16291 Wissenschaftlicher Artikel Lorenzin, Francesca; Benary, Uwe; Baluapuri, Apoorva; Walz, Susanne; Jung, Lisa Anna; von Eyss, Björn; Kisker, Caroline; Wolf, Jana; Eilers, Martin; Wolf, Elmar Different promoter affinities account for specificity in MYC-dependent gene regulation Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells. 2016 e15161 eLife 5 urn:nbn:de:bvb:20-opus-162913 10.7554/eLife.15161 Theodor-Boveri-Institut für Biowissenschaften OPUS4-17107 Wissenschaftlicher Artikel Fischer, Annette; Harrison, Kelly S; Ramirez, Yesid; Auer, Daniela; Chowdhury, Suvagata Roy; Prusty, Bhupesh K; Sauer, Florian; Dimond, Zoe; Kisker, Caroline; Hefty, P Scott; Rudel, Thomas Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense. 2017 eLife 6 e21465 urn:nbn:de:bvb:20-opus-171073 10.7554/eLife.21465 Theodor-Boveri-Institut für Biowissenschaften OPUS4-26164 Wissenschaftlicher Artikel Koelmel, Wolfgang; Kuper, Jochen; Kisker, Caroline Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a "quick-soak"-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy. 2021 17038 Scientific Reports 11 1 urn:nbn:de:bvb:20-opus-261644 10.1038/s41598-021-95186-1 Rudolf-Virchow-Zentrum