DNA mismatch repair protects the genome from oxygen-induced replicative mutagenesis.

DNA mismatch repair (MMR) corrects mismatched DNA bases arising from multiple sources including polymerase errors and base damage. By detecting spontaneous mutagenesis using whole genome sequencing of cultured MMR deficient human cell lines, we show that a primary role of MMR is the repair of oxygen-induced mismatches. We found an approximately twofold higher mutation rate in MSH6 deficient DLD-1 cells or MHL1 deficient HCT116 cells exposed to atmospheric conditions as opposed to mild hypoxia, which correlated with oxidant levels measured using electron paramagnetic resonance spectroscopy. The oxygen-induced mutations were dominated by T to C base substitutions and single T deletions found primarily on the lagging strand. A broad sequence context preference, dependence on replication timing and a lack of transcriptional strand bias further suggested that oxygen-induced mutations arise from polymerase errors rather than oxidative base damage. We defined separate low and high oxygen-specific MMR deficiency mutation signatures common to the two cell lines and showed that the effect of oxygen is observable in MMR deficient cancer genomes, where it best correlates with the contribution of mutation signature SBS21. Our results imply that MMR corrects oxygen-induced genomic mismatches introduced by a replicative process in proliferating cells.

A comparative analysis of the mutagenicity of platinum-containing chemotherapeutic agents reveals direct and indirect mutagenic mechanisms.

Platinum-based drugs are a mainstay of cancer chemotherapy. However, their mutagenic effect can increase tumour heterogeneity, contribute to the evolution of treatment resistance and also induce secondary malignancies. We coupled whole genome sequencing with phenotypic investigations on two cell line models to compare the magnitude and examine the mechanism of mutagenicity of cisplatin, carboplatin and oxaliplatin. Cisplatin induced significantly more base substitution mutations than carboplatin or oxaliplatin when used at equitoxic concentrations on human TK6 or chicken DT40 cells, and also induced the highest number of short insertions and deletions. The analysis of base substitution spectra revealed that all three tested platinum drugs elicit both a direct mutagenic effect at purine dinucleotides, and an indirect effect of accelerating endogenous mutagenic processes, whereas the direct mutagenic effect appeared to correlate with the level of DNA damage caused as assessed through histone H2AX phosphorylation and single-cell agarose gel electrophoresis, the indirect mutagenic effects were equal. The different mutagenicity and DNA-damaging effect of equitoxic platinum drug treatments suggest that DNA damage independent mechanisms significantly contribute to their cytotoxicity. Thus, the comparatively high mutagenicity of cisplatin should be taken into account in the design of chemotherapeutic regimens.

A study on the secondary structure of the metalloregulatory protein CueR: effect of pH, metal ions and DNA.

The response of CueR towards environmental changes in solution was investigated. CueR is a bacterial metal ion selective transcriptional metalloregulator protein, which controls the concentration of copper ions in the cell. Although several articles have been devoted to the discussion of the structural and functional features of this protein, CueR has not previously been extensively characterized in solution. Here, we studied the effect of change in pH, temperature, and the presence of specific or non-specific binding partners on the secondary structure of CueR with circular dichroism (CD) spectroscopy. A rather peculiar reversible pH-dependent secondary structure transformation was observed, elucidated and supplemented with pKa estimation by PROPKA and CpHMD simulations suggesting an important role of His(76) and His(94) in this process. CD experiments revealed that the presence of DNA prevents this structural switch, suggesting that DNA locks CueR in the α-helical-rich form. In contrast to the non-cognate metal ions HgII, CdII and ZnII, the presence of the cognate AgI ion affects the secondary structure of CueR, most probably by stabilizing the metal ion and DNA-binding domains of the protein.

The genomic imprint of cancer therapies helps timing the formation of metastases.

A retrospective determination of the time of metastasis formation is essential for a better understanding of the evolution of oligometastatic cancer. This study was based on the hypothesis that genomic alterations induced by cancer therapies could be used to determine the temporal order of the treatment and the formation of metastases. We analysed the whole genome sequence of a primary tumour sample and three metastatic sites derived from autopsy samples from a young never-smoker lung adenocarcinoma patient with an activating EGFR mutation. Mutation detection methods were refined to accurately detect and distinguish clonal and subclonal mutations. In comparison to a panel of samples from untreated smoker or never-smoker patients, we showed that the mutagenic effect of cisplatin treatment could be specifically detected from the base substitution mutations. Metastases that arose before or after chemotherapeutic treatment could be distinguished based on the allele frequency of cisplatin-induced dinucleotide mutations. In addition, genomic rearrangements and late amplification of the EGFR gene likely induced by afatinib treatment following the acquisition of a T790M gefitinib resistance mutation provided further evidence to tie the time of metastasis formation to treatment history. The established analysis pipeline for the detection of treatment-derived mutations allows the drawing of tumour evolutionary paths based on genomic data, showing that metastases may be seeded well before they become detectable by clinical imaging.

Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease.

Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation circular dichroism spectroscopy, and nano-electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting of three (regulator, DNA-binding, and nuclease) units, rather than simple chimera. The optimisation of such ANs could lead to safe gene editing enzymes.

Abnormal galactosylation of immunoglobulin G in cerebrospinal fluid of multiple sclerosis patients.

BACKGROUND: Glycosylation alterations have been associated with the development of several human diseases and their animal models, including multiple sclerosis. OBJECTIVES: We aimed to determine whether immunoglobulin G galactosylation might be changed in multiple sclerosis. METHODS: Immunoglobulin G was isolated from serum and cerebrospinal fluid of patients with multiple sclerosis or viral meningitis and control patients without history of inflammatory or autoimmune disease. A lectin-based assay was used to investigate potential galactosylation modifications of immunoglobulin G. RESULTS AND CONCLUSION: Galactosylation of immunoglobulin G isolated from cerebrospinal fluid of control patients was found to be age- and gender-dependent. In addition, immunoglobulin G galactosylation was significantly altered in cerebrospinal fluid but not in serum of multiple sclerosis patients. Furthermore, this modification was correlated with an active progression of multiple sclerosis. Finally, the loss of galactosyl moieties was not simply associated with inflammation as no such change was detected in viral meningitis patients characterized by brain inflammation.

Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease.

The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn(2+)-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn(2+) binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.

Design of a colicin E7 based chimeric zinc-finger nuclease.

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity-offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

The mutagenic consequences of defective DNA repair.

Multiple separate repair mechanisms safeguard the genome against various types of DNA damage, and their failure can increase the rate of spontaneous mutagenesis. The malfunction of distinct repair mechanisms leads to genomic instability through different mutagenic processes. For example, defective mismatch repair causes high base substitution rates and microsatellite instability, whereas homologous recombination deficiency is characteristically associated with deletions and chromosome instability. This review presents a comprehensive collection of all mutagenic phenotypes associated with the loss of each DNA repair mechanism, drawing on data from a variety of model organisms and mutagenesis assays, and placing greatest emphasis on systematic analyses of human cancer datasets. We describe the latest theories on the mechanism of each mutagenic process, often explained by reliance on an alternative repair pathway or the error-prone replication of unrepaired, damaged DNA. Aided by the concept of mutational signatures, the genomic phenotypes can be used in cancer diagnosis to identify defective DNA repair pathways.

Comprehensive investigation of the mutagenic potential of six pesticides classified by IARC as probably carcinogenic to humans.

Pesticides are significant environmental pollutants, and many of them possess mutagenic potential, which is closely linked to carcinogenesis. Here we tested the mutagenicity of all six pesticides classified probably carcinogenic (Group 2A) by the International Agency of Research on Cancer: 4,4'-DDT, captafol, dieldrin, diazinon, glyphosate and malathion. Whole genome sequencing of TK6 human lymphoblastoid cell clones following 30-day exposure at subtoxic concentrations revealed a clear mutagenic effect of treatment with captafol or malathion when added at 200 nM or 100 µM initial concentrations, respectively. Each pesticide induced a specific base substitution mutational signature: captafol increased C to A mutations primarily, while malathion induced mostly C to T mutations. 4,4'-DDT, dieldrin, diazinon and glyphosate were not mutagenic. Whereas captafol induced chromosomal instability, H2A.X phosphorylation and cell cycle arrest in G2/M phase, all indicating DNA damage, malathion did not induce DNA damage markers or cell cycle alterations despite its mutagenic effect. Hypersensitivity of REV1 and XPA mutant DT40 chicken cell lines suggests that captafol induces DNA adducts that are bypassed by translesion DNA synthesis and are targets for nucleotide excision repair. The experimentally identified mutational signatures of captafol and malathion could shed light on the mechanism of action of these compounds. The signatures are potentially suitable for detecting past exposure in tumour samples, but the reanalysis of large cancer genome databases did not reveal any evidence of captafol or malathion exposure.

The role of the N-terminal loop in the function of the colicin E7 nuclease domain.

Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX(14)NX(8)HX(3)H ßßα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease domain of ColE7 (NColE7, 446-576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7. The effect of the N-terminal truncation on the Zn(2+) ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement was simulated by molecular dynamics. Semiempirical quantum chemical calculations were performed to obtain better insight into the structure of the active centre. The longer protein interacted with both Zn(2+) ion and DNA more strongly than its shorter counterpart. The results were explained by the structural stabilization effect of the N-terminal amino acids on the catalytic centre. In agreement with this, the absence of the N-terminal sequences resulted in significantly increased movement of the backbone atoms compared with that in the native NColE7: in ΔN25-NColE7 the amino acid strings between residues 485-487, 511-515 and 570-571, and in ΔN4-NColE7 those between residues 467-468, 530-535 and 570-571.

Spontaneous mutagenesis in human cells is controlled by REV1-Polymerase ζ and PRIMPOL.

Translesion DNA synthesis (TLS) facilitates replication over damaged or difficult-to-replicate templates by employing specialized DNA polymerases. We investigate the effect on spontaneous mutagenesis of three main TLS control mechanisms: REV1 and PCNA ubiquitylation that recruit TLS polymerases and PRIMPOL that creates post-replicative gaps. Using whole-genome sequencing of cultured human RPE-1 cell clones, we find that REV1 and Polymerase ζ are wholly responsible for one component of base substitution mutagenesis that resembles homologous recombination deficiency, whereas the remaining component that approximates oxidative mutagenesis is reduced in PRIMPOL-/- cells. Small deletions in short repeats appear in REV1-/-PCNAK164R/K164R double mutants, revealing an alternative TLS mechanism. Also, 500-5,000 bp deletions appear in REV1-/- and REV3L-/- mutants, and chromosomal instability is detectable in REV1-/-PRIMPOL-/- cells. Our results indicate that TLS protects the genome from deletions and large rearrangements at the expense of being responsible for the majority of spontaneous base substitutions.

Prospectively defined patterns of APOBEC3A mutagenesis are prevalent in human cancers.

Mutational signatures defined by single base substitution (SBS) patterns in cancer have elucidated potential mutagenic processes that contribute to malignancy. Two prevalent mutational patterns in human cancers are attributed to the APOBEC3 cytidine deaminase enzymes. Among the seven human APOBEC3 proteins, APOBEC3A is a potent deaminase and proposed driver of cancer mutagenesis. In this study, we prospectively examine genome-wide aberrations by expressing human APOBEC3A in avian DT40 cells. From whole-genome sequencing, we detect hundreds to thousands of base substitutions per genome. The APOBEC3A signature includes widespread cytidine mutations and a unique insertion-deletion (indel) signature consisting largely of cytidine deletions. This multi-dimensional APOBEC3A signature is prevalent in human cancer genomes. Our data further reveal replication-associated mutations, the rate of stem-loop and clustered mutations, and deamination of methylated cytidines. This comprehensive signature of APOBEC3A mutagenesis is a tool for future studies and a potential biomarker for APOBEC3 activity in cancer.

BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1.

Defects in BRCA1, BRCA2 and other genes of the homology-dependent DNA repair (HR) pathway cause an elevated rate of mutagenesis, eliciting specific mutation patterns including COSMIC signature SBS3. Using genome sequencing of knock-out cell lines we show that Y family translesion synthesis (TLS) polymerases contribute to the spontaneous generation of base substitution and short insertion/deletion mutations in BRCA1 deficient cells, and that TLS on DNA adducts is increased in BRCA1 and BRCA2 mutants. The inactivation of 53BP1 in BRCA1 mutant cells markedly reduces TLS-specific mutagenesis, and rescues the deficiency of template switch-mediated gene conversions in the immunoglobulin V locus of BRCA1 mutant chicken DT40 cells. 53BP1 also promotes TLS in human cellular extracts in vitro. Our results show that HR deficiency-specific mutagenesis is largely caused by TLS, and suggest a function for 53BP1 in regulating the choice between TLS and error-free template switching in replicative DNA damage bypass.

Decreased pH in the aging brain and Alzheimer's disease.

Using publicly available data sets, we compared pH in the human brain and the cerebrospinal fluid (CSF) of postmortem control and Alzheimer's disease cases. We further investigated the effects of long-term acidosis in vivo in the APP-PS1 mouse model of Alzheimer's disease. We finally examined in vitro whether low pH exposure could modulate the release of proinflammatory cytokines and the uptake of amyloid beta by microglia. In the human brain, pH decreased with aging. Similarly, we observed a reduction of pH in the brain of C57BL/6 mice with age. In addition, independent database analyses revealed that postmortem brain and CSF pH is further reduced in Alzheimer's disease cases compared with controls. Moreover, in vivo experiments showed that low pH CSF infusion increased amyloid beta plaque load in APP-PS1 mice. We further observed that mild acidosis reduced the amyloid beta 42-induced release of tumor necrosis factor-alpha by microglia and their capacity to uptake this peptide. Brain acidosis is associated with aging and might affect pathophysiological processes such as amyloid beta aggregation or inflammation in Alzheimer's disease.

Two main mutational processes operate in the absence of DNA mismatch repair.

The analysis of tumour genome sequences has demonstrated high rates of base substitution mutagenesis upon the inactivation of DNA mismatch repair (MMR), and the resulting somatic mutations in MMR deficient tumours appear to significantly enhance the response to immune therapy. A handful of different algorithmically derived base substitution mutation signatures have been attributed to MMR deficiency in tumour somatic mutation datasets. In contrast, mutation data obtained from whole genome sequences of isogenic wild type and MMR deficient cell lines in this study, as well as from published sources, show a more uniform experimental mutation spectrum of MMR deficiency. In order to resolve this discrepancy, we reanalysed mutation data from MMR deficient tumour whole exome and whole genome sequences. We derived two base substitution signatures using non-negative matrix factorisation, which together adequately describe mutagenesis in all tumour and cell line samples. The two new signatures broadly resemble COSMIC signatures 6 and 20, but perform better than existing COSMIC signatures at identifying MMR deficient tumours in mutation signature deconstruction. We show that the contribution of the two identified signatures, one of which is dominated by C to T mutations at CpG sites, is biased by the different sequence composition of the exome and the whole genome. We further show that the identity of the inactivated MMR gene, the tissue type, the mutational burden or the patient's age does not influence the mutation spectrum, but that a tendency for a greater contribution by the CpG mutational process is observed in tumours as compared to cultured cells. Our analysis suggest that two separable mutational processes operate in the genomes of MMR deficient cells.

Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells.

Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and a predictor of recurrent malignant disease. Understanding how human ABCG2 expression is modulated by pharmacotherapy is crucial in guiding therapeutic recommendations and may aid rational drug development. Genome edited reporter cells are useful in investigating gene regulation and visualizing protein activity in live cells but require precise targeting to preserve native regulatory regions. Here, we describe a fluorescent reporter assay that allows the noninvasive assessment of ABCG2 regulation in human lung adenocarcinoma cells. Using CRISPR-Cas9 gene editing coupled with homology-directed repair, we targeted an EGFP coding sequence to the translational start site of ABCG2, generating ABCG2 knock-out and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that ABCG2 is upregulated by a number of anti-cancer medications, HDAC inhibitors, hypoxia-mimicking agents and glucocorticoids, supporting a model in which ABCG2 is under the control of a general stress response. To our knowledge, this is the first description of a fluorescent reporter assay system designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may guide therapy recommendations and aid rational drug design.

Correlation of homologous recombination deficiency induced mutational signatures with sensitivity to PARP inhibitors and cytotoxic agents.

BACKGROUND: Homologous recombination (HR) repair deficiency arising from defects in BRCA1 or BRCA2 is associated with characteristic patterns of somatic mutations. In this genetic study, we ask whether inactivating mutations in further genes of the HR pathway or the DNA damage checkpoint also give rise to somatic mutation patterns that can be used for treatment prediction. RESULTS: Using whole genome sequencing of an isogenic knockout cell line panel, we find a universal HR deficiency-specific base substitution signature that is similar to COSMIC signature 3. In contrast, we detect different deletion phenotypes corresponding to specific HR mutants. The inactivation of BRCA2 or PALB2 leads to larger deletions, typically with microhomology, when compared to the disruption of BRCA1, RAD51 paralogs, or RAD54. Comparison with the deletion spectrum of Cas9 cut sites suggests that most spontaneously arising genomic deletions are not the consequence of double-strand breaks. Surprisingly, the inactivation of checkpoint kinases ATM and CHK2 has no mutagenic consequences. Analysis of tumor exomes with biallelic inactivating mutations in the investigated genes confirms the validity of the cell line models. We present a comprehensive analysis of sensitivity of the investigated mutants to 13 therapeutic agents for the purpose of correlating genomic mutagenic phenotypes with drug sensitivity. CONCLUSION: Our results suggest that no single genomic mutational class shows perfect correlation with sensitivity to common treatments, but the contribution of COSMIC signature 3 to base substitutions, or a combined measure of different features, may be reasonably good at predicting platinum and PARP inhibitor sensitivity.

Analysis of the vasculature by immunohistochemistry in paraffin-embedded brains.

The brain vasculature can be investigated in different ways ranging from in vivo to biochemical analysis. Immunohistochemistry is a simple and powerful technique that can also be applied to archival tissues. However, staining of brain vessels on paraffin sections has been challenging. In this study, we developed an optimized method that can be used in paraffin-embedded mouse and human brain tissues derived from healthy controls and neurological disorders such as Alzheimer's disease. We subsequently showed that this method is fully compatible with the detection of glial cells and key markers of Alzheimer's disease including amyloid beta and phosphorylated Tau protein. Furthermore, we observed that the length of microvasculature in hippocampus of TgCRND8 Alzheimer's disease mouse model is reduced, which is correlated with the decreased blood flow in hippocampus as determined by arterial spin labeling perfusion magnetic resonance imaging. Finally, we determined that the microvasculature length in two other Alzheimer's disease mouse models, APP and PS1 double-transgenic mice and P301S Tau-transgenic mice, is also shortened in the dentate gyrus. Thus, we have established a new, simple and robust method to characterize the brain vasculature in the mouse and human brain.

[Granulomatous hepatitis successfully treated with ursodeoxycholic acid]. / Ursodeoxycholsavval sikeresen kezelt granulomás hepatitis.

The etiology of granulomatous hepatitis is extremely extensive for which the granulomatous reaction induced hepatic inflammation is characteristic. In the majority of cases it is part of a systemic disease but sometimes there is primer hepatic lesion in the background. The 21 year-old Caucasian female patient was taken to hospital having flu-like symptoms and icterus. While searching for the cause of her cholestatic hepatocellular lesion infections, tumors, storage diseases were excluded. However the root cause could not be identified. The histology showed granulomatous reaction, inflammation, intrahepatic bile duct proliferation and micronodular cirrhosis in the liver tissue. Taken it into consideration together with the the clinical picture and the laboratory findings primer sclerosing cholangitis was rendered probable. Her state deteriorated and even the necessity of liver transplantation emerged. The patient was treated with ursodeoxycholic acid which resulted in remission lasting for years now. Two years after the onset of the hepatic disease pancolitis ulcerosa with affection of the ileum terminalis was found in the patient which supports the diagnosis of primary sclerosing cholangitis.