@article{SchmittKuperEliasetal.2014, author = {Schmitt, Dominik R. and Kuper, Jochen and Elias, Agnes and Kisker, Caroline}, title = {The Structure of the TFIIH p34 Subunit Reveals a Von Willebrand Factor A Like Fold}, series = {PLoS ONE}, volume = {9}, journal = {PLoS ONE}, number = {7}, issn = {1932-6203}, doi = {10.1371/journal.pone.0102389}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119471}, pages = {e102389}, year = {2014}, abstract = {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.}, language = {en} } @article{MeirMaurusKuperetal.2021, author = {Meir, Michael and Maurus, Katja and Kuper, Jochen and Hankir, Mohammed and Wardelmann, Eva and Rosenwald, Andreas and Germer, Christoph-Thomas and Wiegering, Armin}, title = {The novel KIT exon 11 germline mutation K558N is associated with gastrointestinal stromal tumor, mastocytosis, and seminoma development}, series = {Genes, Chromosomes \& Cancer}, volume = {60}, journal = {Genes, Chromosomes \& Cancer}, number = {12}, doi = {10.1002/gcc.22988}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257476}, pages = {827-832}, year = {2021}, abstract = {Familial gastrointestinal stromal tumors (GIST) are dominant genetic disorders that are caused by germline mutations of the type III receptor tyrosine kinase KIT. While sporadic mutations are frequently found in mastocytosis and GISTs, germline mutations of KIT have only been described in 39 families until now. We detected a novel germline mutation of KIT in exon 11 (p.Lys-558-Asn; K558N) in a patient from a kindred with several GISTs harboring different secondary somatic KIT mutations. Structural analysis suggests that the primary germline mutation alone is not sufficient to release the autoinhibitory region of KIT located in the transmembrane domain. Instead, the KIT kinase module becomes constitutively activated when K558N combines with different secondary somatic mutations. The identical germline mutation in combination with an additional somatic KIT mutation was detected in a second patient of the kindred with seminoma while a third patient within the family had a cutaneous mastocytosis. These findings suggest that the K558N mutation interferes with the juxtamembranous part of KIT, since seminoma and mastocystosis are usually not associated with exon 11 mutations.}, language = {en} } @article{RaduSchoenwetterBraunetal.2017, author = {Radu, Laura and Schoenwetter, Elisabeth and Braun, Cathy and Marcoux, Julien and Koelmel, Wolfgang and Schmitt, Dominik R. and Kuper, Jochen and Cianf{\´e}rani, Sarah and Egly, Jean M. and Poterszman, Arnaud and Kisker, Caroline}, title = {The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH}, series = {Nucleic Acids Research}, volume = {45}, journal = {Nucleic Acids Research}, number = {18}, doi = {10.1093/nar/gkx743}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170173}, pages = {10872-10883}, year = {2017}, abstract = {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.}, language = {en} } @article{PetrusevaNaumenkoKuperetal.2021, author = {Petruseva, Irina and Naumenko, Natalia and Kuper, Jochen and Anarbaev, Rashid and Kappenberger, Jeannette and Kisker, Caroline and Lavrik, Olga}, title = {The Interaction Efficiency of XPD-p44 With Bulky DNA Damages Depends on the Structure of the Damage}, series = {Frontiers in Cell and Developmental Biology}, volume = {9}, journal = {Frontiers in Cell and Developmental Biology}, issn = {2296-634X}, doi = {10.3389/fcell.2021.617160}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231806}, year = {2021}, abstract = {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.}, language = {en} } @unpublished{NeitzBessiKuperetal.2023, author = {Neitz, Hermann and Bessi, Irene and Kuper, Jochen and Kisker, Caroline and H{\"o}bartner, Claudia}, title = {Programmable DNA interstrand crosslinking by alkene-alkyne [2+2] photocycloaddition}, series = {Journal of the American Chemical Society}, journal = {Journal of the American Chemical Society}, edition = {submitted version}, doi = {10.1021/jacs.3c01611}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311822}, year = {2023}, abstract = {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.}, language = {en} } @article{PeissertSauerGrabarczyketal.2020, author = {Peissert, Stefan and Sauer, Florian and Grabarczyk, Daniel B. and Braun, Cathy and Sander, Gudrun and Poterszman, Arnaud and Egly, Jean-Marc and Kuper, Jochen and Kisker, Caroline}, title = {In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair}, series = {Nature Communications}, volume = {11}, journal = {Nature Communications}, number = {1}, doi = {10.1038/s41467-020-15241-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229857}, year = {2020}, abstract = {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.}, language = {en} } @article{KhanSuhasiniBanerjeeetal.2014, author = {Khan, Irfan and Suhasini, Avvaru N. and Banerjee, Taraswi and Sommers, Joshua A. and Kaplan, Daniel L. and Kuper, Jochen and Kisker, Caroline and Brosh, Jr., Robert M.}, title = {Impact of Age-Associated Cyclopurine Lesions on DNA Repair Helicases}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {11}, doi = {10.1371/journal.pone.0113293}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114635}, pages = {e113293}, year = {2014}, abstract = {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.}, language = {en} } @article{ReuterJaeckelsKneitzetal.2019, author = {Reuter, Isabel and J{\"a}ckels, Jana and Kneitz, Susanne and Kuper, Jochen and Lesch, Klaus-Peter and Lillesaar, Christina}, title = {Fgf3 is crucial for the generation of monoaminergic cerebrospinal fluid contacting cells in zebrafish}, series = {Biology Open}, volume = {8}, journal = {Biology Open}, doi = {10.1242/bio.040683}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200749}, pages = {bio040683}, year = {2019}, abstract = {In most vertebrates, including zebrafish, the hypothalamic serotonergic cerebrospinal fluid-contacting (CSF-c) cells constitute a prominent population. In contrast to the hindbrain serotonergic neurons, little is known about the development and function of these cells. Here, we identify fibroblast growth factor (Fgf)3 as the main Fgf ligand controlling the ontogeny of serotonergic CSF-c cells. We show that fgf3 positively regulates the number of serotonergic CSF-c cells, as well as a subset of dopaminergic and neuroendocrine cells in the posterior hypothalamus via control of proliferation and cell survival. Further, expression of the ETS-domain transcription factor etv5b is downregulated after fgf3 impairment. Previous findings identified etv5b as critical for the proliferation of serotonergic progenitors in the hypothalamus, and therefore we now suggest that Fgf3 acts via etv5b during early development to ultimately control the number of mature serotonergic CSF-c cells. Moreover, our analysis of the developing hypothalamic transcriptome shows that the expression of fgf3 is upregulated upon fgf3 loss-of-function, suggesting activation of a self-compensatory mechanism. Together, these results highlight Fgf3 in a novel context as part of a signalling pathway of critical importance for hypothalamic development.}, language = {en} } @article{RohlederHuangXueetal.2016, author = {Rohleder, Florian and Huang, Jing and Xue, Yutong and Kuper, Jochen and Round, Adam and Seidman, Michael and Wang, Weidong and Kisker, Caroline}, title = {FANCM interacts with PCNA to promote replication traverse of DNA interstrand crosslinks}, series = {Nucleic Acids Research}, volume = {44}, journal = {Nucleic Acids Research}, number = {7}, doi = {10.1093/nar/gkw037}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175401}, pages = {3219-3232}, year = {2016}, abstract = {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.}, language = {en} } @article{KoelmelKuperKisker2021, author = {Koelmel, Wolfgang and Kuper, Jochen and Kisker, Caroline}, title = {Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem}, series = {Scientific Reports}, volume = {11}, journal = {Scientific Reports}, number = {1}, doi = {10.1038/s41598-021-95186-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261644}, pages = {17038}, year = {2021}, abstract = {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.}, language = {en} }