Journal: Nucleic Acids Research

Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

doi: 10.1093/nar/gks802

Figure Lengend Snippet: Hybrid G-quadruplexes form during transcription and are dependent on CSB II. ( A ) In vitro transcription using the mitochondrial transcription apparatus on supercoiled templates containing either wildtype or mutant CSB II. Transcription reactions were divided into three parts, and either not treated or treated with RNase A and/or hRNaseH1, as indicated. RNase A-treatment reveals a non-degradable product of 45–50 bp (marked by asterisks) that is dependent on the CSB II sequence and Hoogsteen base pairing. ( B ) The labeled CSB II RNA oligonucleotide was allowed to form G-quadruplexes alone (lanes 1–3), with wildtype CSB II DNA oligonucleotide of identical sequence (lanes 4–6), or with a reverse complement DNA oligonucleotide (lanes 7–9), and then subjected to treatment with RNase A or hRNaseH1 in order to show that the G-quadruplex hybrid is resistant to hRNaseH1 (lane 6). The Watson–Crick basepaired hybrid with reverse complement DNA was degraded by hRNaseH1 as expected (lane 9). M, marker lane. ( C ) Transcription on templates encompassing base positions 1–477 of human mtDNA and containing either dGTP or 7-deaza-dGTP (lanes 1 and 2). After transcription, part of each reaction was treated with RNaseA (lanes 3 and 4). The RNaseA-resistant species of ∼50 bp (indicated by black bar) are absent when G-quadruplex formation with the template is eliminated (lane 3). M, marker lane.

Article Snippet: When indicated, 7-deaza-dGTP (New England Biolabs) replaced dGTP in the PCR reactions.

Techniques: In Vitro, Mutagenesis, Sequencing, Labeling, Marker

Journal: Nucleic Acids Research

Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

doi: 10.1093/nar/gks802

Figure Lengend Snippet: A longer R-loop can form during transcription of human mtDNA with phage T7 RNA polymerase. ( A ) Transcription of a human mtDNA template containing wildtype or mutant CSB II with T7 RNA pol in the presence of either GTP (lanes 1–3 and 7–9) or 7-deaza-GTP (lanes 4–6 and 10–12). A hybrid species of ∼120 bp is revealed upon RNase A treatment (lane 2) and is sensitive to hRNaseH1 (lane 3). This longer hybrid is dependent on CSB II and not observed in the presence of 7-deaza-GTP (lanes 4–6). ( B ) Schematic presentation of the RNA–DNA hybrid G-quadruplex that forms between the RNA and the non-template DNA strand during transcription of mtDNA. Under some conditions, an extended R-loop may be formed, similar to that reported in ( 22 ).

Article Snippet: When indicated, 7-deaza-dGTP (New England Biolabs) replaced dGTP in the PCR reactions.

Techniques: Mutagenesis

Journal: Nucleic Acids Research

Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

doi: 10.1093/nar/gks802

Figure Lengend Snippet: G-quadruplex formation in nascent RNA, but not in template DNA, causes premature termination of transcription. In vitro transcription with the mitochondrial transcription apparatus was carried out in the presence or absence of 7-deaza-GTP to monitor effects of G-quadruplex formation in RNA. To address the effects of G-quadruplex formation in DNA, the DNA templates used were produced by PCR amplification of positions 1–512 of human mtDNA in the presence or absence of 7-deaza-dGTP.

Article Snippet: When indicated, 7-deaza-dGTP (New England Biolabs) replaced dGTP in the PCR reactions.

Techniques: In Vitro, Produced, Polymerase Chain Reaction, Amplification

Journal: Nucleic Acids Research

Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

doi: 10.1093/nar/gky896

Figure Lengend Snippet: Activity of MTH1 with O6-methyl-dGTP has been conserved through evolution. ( A ) Comparison of activities of MTH1 (NUDT1) (1.5 nM) from different species with 75 μM 8-oxo-dGTP and 75 μM O6-methyl-dGTP (hsNUDT1, human NUDT1; mmNUDT1, mouse NUDT1; rnNUDT1, rat NUDT1; ssNUDT1, pig NUDT1; clNUDT1, dog NUDT1; atNUDX1, Arabidopsis thaliana NUDT1; zfNUDT1, zebrafish MTH1; MutT, E. coli MutT). Reaction was performed in MTH1 reaction buffer and reaction time was 15 min. Formed PPi was detected using PPiLight Inorganic Pyrophosphate Assay kit from Lonza. Data points were recorded in triplicate. ( B ) Ratio between activities with O6-methyl-dGTP and 8-oxo-dGTP for MTH1 from different species.

Article Snippet: We therefore produced N7-methyl-dGTP and tested the ability of MTH1 to catalyze the hydrolysis of N7-methyl-dGTP. dGTP was methylated by incubating 50 mM dGTP (Sigma-Aldrich) at 22°C for 20 h with 100 mM MMS diluted in DMSO, or the same volume DMSO, resulting in a dGTP:MMS ratio of 1:2.

Techniques: Activity Assay, Pyrophosphate Assay

Journal: Nucleic Acids Research

Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

doi: 10.1093/nar/gky896

Figure Lengend Snippet: Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

Article Snippet: We therefore produced N7-methyl-dGTP and tested the ability of MTH1 to catalyze the hydrolysis of N7-methyl-dGTP. dGTP was methylated by incubating 50 mM dGTP (Sigma-Aldrich) at 22°C for 20 h with 100 mM MMS diluted in DMSO, or the same volume DMSO, resulting in a dGTP:MMS ratio of 1:2.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein *

doi: 10.1074/jbc.M114.626275

Figure Lengend Snippet: Binding of mant-deoxyguanine nucleotides to EFL1. Mant fluorescence was excited by FRET from the intrinsic tryptophan residues in EFL1. Concentrations after mixing consisted of 4 μ m EFL1, 50 μ m mant-deoxy-GDP/deoxy-GTP, and 5 m m Mg 2+ as

Article Snippet: The fluorescent mant-derivatives of GDP, GTP, dGDP, dGTP, and GppNHp were obtained from Jena Biosciences (Jena, Germany).

Techniques: Binding Assay, Fluorescence

Journal: Nature

Article Title: Stereospecific targeting of MTH1 by (S)-crizotinib as anticancer strategy

doi: 10.1038/nature13194

Figure Lengend Snippet: MTH1 is the target of SCH51344 a , Representation of the chemical proteomic workflow. b , Sructures of SCH51344 ( 1 ) and the probe used for affinity purification ( 2 ). c , Results from MS-based proteomic affinity purification experiment using SAINT and competition analysis. Data shown are based on two independent experiments for each condition (n = 2/condition), and each replicate was analysed in two technical replicates. d , ITC data for MTH1 with SCH51344. The measured K d was 49 nM (n = 1). e , SCH51344 inhibits hydrolysis of the MTH1 substrates dGTP, 8-oxo-dGTP, and 2-OH-dATP, respectively. Data are shown for two technical replicates ± SEM and representative for at least duplicate experiments (n ≥ 2). f , Silencing of MTH1 by siRNA impairs colony formation of KRAS-positive SW480 (top) and DLD1 (bottom) cells. Data shown as mean ± SEM and images are representative for triplicate experiments (n = 3) (P

Article Snippet: After addition of the substrate dGTP (Fermentas, final concentration 100 μM), 8-oxo-dGTP (TriLink Biotechnologies, final concentration 13.2 μM), or 2-OH-dATP (Jena Bioscience, final concentration 8.3 μM) the generation of pyrophosphate (PPi) as a result of nucleotide triphosphate hydrolysis by MTH1 was monitored over a time course of 15 min using the PPiLight Inorganic Pyrophosphate Assay kit (Lonza Rockland).

Techniques: Affinity Purification, Mass Spectrometry