Publications by authors named "Tobias T Schmidt"

11 Publications

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Telomeres and Cancer: Resolving the Paradox.

Annu Rev Cancer Biol 2021 Mar;5(1):59-77

Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

Decades of study on cell cycle regulation have provided great insight into human cellular life span barriers, as well as their dysregulation during tumorigenesis. Telomeres, the extremities of linear chromosomes, perform an essential role in implementing these proliferative boundaries and preventing the propagation of potentially cancerous cells. The tumor-suppressive function of telomeres relies on their ability to initiate DNA damage signaling pathways and downstream cellular events, ranging from cell cycle perturbation to inflammation and cell death. While the tumor-suppressor role of telomeres is undoubtable, recent advances have pointed to telomeres as a major source of many of the genomic aberrations found in both early- and late-stage cancers, including the most recently discovered mutational phenomenon of chromothripsis. Telomere shortening appears as a double-edged sword that can function in opposing directions in carcinogenesis. This review focuses on the current knowledge of the dual role of telomeres in cancer and suggests a new perspective to reconcile the paradox of telomeres and their implications in cancer etiology.
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http://dx.doi.org/10.1146/annurev-cancerbio-050420-023410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8442540PMC
March 2021

Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function.

Commun Biol 2020 12 10;3(1):751. Epub 2020 Dec 10.

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.

Inactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S. cerevisiae. MLH2 mutations elevated frameshift mutation rates, and caused accumulation of long-lasting nuclear MMR foci. Both aspects of this phenotype were suppressed by mutations predicted to prevent the binding of Mlh2 to DNA. Genetic analysis revealed that mlh2 dominant mutations interfere with both Exonuclease 1 (Exo1)-dependent and Exo1-independent MMR. Lastly, we demonstrate that a homolog mutation in human hPMS1 results in a dominant mutator phenotype. Our data support a model in which yeast Mlh1-Mlh2 or hMLH1-hPMS1 mutant complexes act as roadblocks on DNA preventing MMR, unraveling a novel mechanism that can account for MSI in human cancer.
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http://dx.doi.org/10.1038/s42003-020-01481-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730388PMC
December 2020

Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio.

Nucleic Acids Res 2020 01;48(1):264-277

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg D-69120, Germany.

The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation. Here, we found that Met7-deficient strains show elevated mutation rates, but also increased levels of endogenous DNA damage resulting in gross chromosomal rearrangements (GCRs). Quantification of deoxyribonucleotide (dNTP) pools in cell extracts from met7Δ mutant revealed reductions in dTTP and dGTP that cause a constitutively active DNA damage checkpoint. In addition, we found that the absence of Met7 leads to dUTP accumulation, at levels that allowed its detection in yeast extracts for the first time. Consequently, a high dUTP/dTTP ratio promotes uracil incorporation into DNA, followed by futile repair cycles that compromise both mitochondrial and nuclear DNA integrity. In summary, this work highlights the importance of folate polyglutamylation in the maintenance of nucleotide homeostasis and genome stability.
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http://dx.doi.org/10.1093/nar/gkz1006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145683PMC
January 2020

A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance.

Nucleic Acids Res 2019 01;47(1):237-252

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg D-69120, Germany.

The balance and the overall concentration of intracellular deoxyribonucleoside triphosphates (dNTPs) are important determinants of faithful DNA replication. Despite the established fact that changes in dNTP pools negatively influence DNA replication fidelity, it is not clear why certain dNTP pool alterations are more mutagenic than others. As intracellular dNTP pools are mainly controlled by ribonucleotide reductase (RNR), and given the limited number of eukaryotic RNR mutations characterized so far, we screened for RNR1 mutations causing mutator phenotypes in Saccharomyces cerevisiae. We identified 24 rnr1 mutant alleles resulting in diverse mutator phenotypes linked in most cases to imbalanced dNTPs. Among the identified rnr1 alleles the strongest mutators presented a dNTP imbalance in which three out of the four dNTPs were elevated (dCTP, dTTP and dGTP), particularly if dGTP levels were highly increased. These rnr1 alleles caused growth defects/lethality in DNA replication fidelity-compromised backgrounds, and caused strong mutator phenotypes even in the presence of functional DNA polymerases and mismatch repair. In summary, this study pinpoints key residues that contribute to allosteric regulation of RNR's overall activity or substrate specificity. We propose a model that distinguishes between different dNTP pool alterations and provides a mechanistic explanation why certain dNTP imbalances are particularly detrimental.
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http://dx.doi.org/10.1093/nar/gky1154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326808PMC
January 2019

Alterations in cellular metabolism triggered by or inactivation cause imbalanced dNTP pools and increased mutagenesis.

Proc Natl Acad Sci U S A 2017 05 17;114(22):E4442-E4451. Epub 2017 Apr 17.

German Cancer Research Center, 69120 Heidelberg, Germany;

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (, , and ) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.
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http://dx.doi.org/10.1073/pnas.1618714114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465912PMC
May 2017

Visualization of mismatch repair complexes using fluorescence microscopy.

DNA Repair (Amst) 2016 Feb 12;38:58-67. Epub 2015 Dec 12.

German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany. Electronic address:

DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients. During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5' and 3' nick-directed MMR. However, several mechanistic aspects of the MMR reaction remain not fully understood, therefore alternative approaches and further investigations are needed. Recently, the use of imaging techniques and, more specifically, visualization of MMR components in living cells, has broadened our mechanistic understanding of the repair reaction providing more detailed information about the spatio-temporal organization of MMR in vivo. In this review we would like to comment on mechanistic aspects of the MMR reaction in light of these and other recent findings. Moreover, we will discuss the current limitations and provide future perspectives regarding imaging of mismatch repair components in diverse organisms.
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http://dx.doi.org/10.1016/j.dnarep.2015.11.014DOI Listing
February 2016

Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction.

Chembiochem 2015 May 5;16(8):1158-62. Epub 2015 May 5.

Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany).

Inverse-electron-demand Diels-Alder cycloaddition (DAinv ) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA(W37V) ), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DAinv  . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13-residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two-step labeling strategy to label proteins in cell lysates in a site-specific manner and performed cell-surface labeling on living cells.
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http://dx.doi.org/10.1002/cbic.201500042DOI Listing
May 2015

New insights into the mechanism of DNA mismatch repair.

Chromosoma 2015 Dec 11;124(4):443-62. Epub 2015 Apr 11.

German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.

The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by up-to-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR.
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http://dx.doi.org/10.1007/s00412-015-0514-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600670PMC
December 2015

Two-step protein labeling utilizing lipoic acid ligase and Sonogashira cross-coupling.

Bioconjug Chem 2014 Sep 27;25(9):1632-7. Epub 2014 Aug 27.

Institute for Pharmacy and Molecular Biotechnology, Heidelberg University , Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.

Labeling proteins in their natural settings with fluorescent proteins or protein tags often leads to problems. Despite the high specificity, these methods influence the natural functions due to the rather large size of the proteins used. Here we present a two-step labeling procedure for the attachment of various fluorescent probes to a small peptide sequence (13 amino acids) using enzyme-mediated peptide labeling in combination with palladium-catalyzed Sonogashira cross-coupling. We identified p-iodophenyl derivatives from a small library that can be covalently attached to a lysine residue within a specific 13-amino-acid peptide sequence by Escherichia coli lipoic acid ligase A (LplA). The derivatization with p-iodophenyl subsequently served as a reactive handle for bioorthogonal transition metal-catalyzed Sonogashira cross-coupling with alkyne-functionalized fluorophores on both the peptide as well as on the protein level. Our two-step labeling strategy combines high selectivity of enzyme-mediated labeling with the chemoselectivity of palladium-catalyzed Sonogashira cross-coupling.
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http://dx.doi.org/10.1021/bc500349hDOI Listing
September 2014

In-vitro evaluation of chronic alcohol effects on expression of drug-metabolizing and drug-transporting proteins.

J Pharm Pharmacol 2013 Oct 30;65(10):1518-25. Epub 2013 Jul 30.

Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Heidelberg, Germany.

Objectives: In alcoholics without alcoholic liver disease, boosted drug elimination has been reported. However, mechanistic explanations for this phenomenon remain uncertain. In particular, data on the potential role of drug transporters are sparse.

Methods: Using a well-established in-vitro model for induction of human drug-metabolizing and drug-transporting proteins, we evaluated the potency of ethanol and the major fermentation side-product isopentanol to alter expression and function of these proteins by quantitative real-time polymerase chain reaction, Western blotting and flow cytometry. P-glycoprotein (Pgp)-inhibiting properties of ethanol and isopentanol were investigated via calcein extrusion assay.

Key Findings: Ethanol and isopentanol significantly changed expression levels of drug-metabolizing and drug-transporting proteins that normalized within 2 weeks upon withdrawal. Cytochrome P-450 2C19 and Pgp were most strongly induced. Ethanol-induced Pgp at the messenger RNA (mRNA) (twofold to eightfold) and protein level (twofold), but not at the functional level. Both compounds did not inhibit Pgp.

Conclusions: Ethanol is demonstrated to increase mRNA and protein expression of human drug transporters such as Pgp in vitro. Withdrawal of ethanol exposure causes return to non-induced conditions within weeks. Functional consequences of increased Pgp expression in alcoholics need to be evaluated by clinical trials applying selective Pgp substrates such as digoxin.
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http://dx.doi.org/10.1111/jphp.12124DOI Listing
October 2013

Proteoglycans act as cellular hepatitis delta virus attachment receptors.

PLoS One 2013 7;8(3):e58340. Epub 2013 Mar 7.

Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Baden-Württemberg, Germany.

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058340PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591349PMC
September 2013
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