aag  (New England Biolabs)

Journal: Chemical Science

Article Title: Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e

doi: 10.1039/c7sc04296e

Figure Lengend Snippet: (A) Variance of the initial velocity in response to various concentrations of the Cy3-labeled molecular beacon substrate. The concentration of hOGG1 is 0.1 U μL –1 and the concentration of APE1 is 0.1 U μL –1 . (B) Variance of the initial velocity in response to various concentrations of the Cy5-labeled molecular beacon substrate. The concentration of hAAG is 0.1 U μL –1 and the concentration of APE1 is 0.1 U μL –1 . The error bars represent the standard deviations of the three experiments.

Article Snippet: Formation of the molecular beacons and the enzyme reactions Firstly, 6 μM hOGG1 substrate, 6 μM hAAG substrate and the mixture of 6 μM hOGG1 substrate and 6 μM hAAG substrate were incubated in 1× NEBuffer 2 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) at 95 °C for 5 min, respectively.

Techniques: Labeling, Concentration Assay

Journal: Chemical Science

Article Title: Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e

doi: 10.1039/c7sc04296e

Figure Lengend Snippet: Variance of the relative catalytic activity with different concentrations of Cd 2+ for hOGG1 alone (black line), hOGG1 + APE1 (red line) and hAAG + APE1 (blue line). The Cy3-labeled molecular beacon (0.3 μM), the Cy5-labeled molecular beacon (0.3 μM), hOGG1 (0.1 U μL –1 ), hAAG (0.1 U μL –1 ) and APE1 (0.1 U μL –1 ) were used in this research. The error bars represent the standard deviations of the three experiments.

Article Snippet: Formation of the molecular beacons and the enzyme reactions Firstly, 6 μM hOGG1 substrate, 6 μM hAAG substrate and the mixture of 6 μM hOGG1 substrate and 6 μM hAAG substrate were incubated in 1× NEBuffer 2 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) at 95 °C for 5 min, respectively.

Techniques: Activity Assay, Labeling

Journal: Chemical Science

Article Title: Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e

doi: 10.1039/c7sc04296e

Figure Lengend Snippet: (A) PAGE analysis of the hOGG1-mediated cleavage of the Cy3-labeled molecular beacon (lanes 1–4) and the hAAG-mediated cleavage of the Cy5-labeled molecular beacon (lanes 5–8) with SYBR Gold as the indicator. (B) PAGE analysis of the hOGG1-mediated cleavage of the Cy3-labeled molecular beacon and the hAAG-mediated cleavage of the Cy5-labeled molecular beacon by excitation of Cy3 and Cy5. The green color indicates the Cy3-labeled DNA fragment in the presence of hOGG1 (lane 2) and the red color indicates the Cy5-labeled DNA fragment in the presence of hAAG (lane 4). (C) Fluorescence measurements of the hOGG1-mediated cleavage of the Cy3-labeled molecular beacon in the absence (black line) and presence (green line) of hOGG1. (D) Fluorescence measurements of the hAAG-mediated cleavage of the Cy5-labeled molecular beacon in the absence (blue line) and presence (red line) of hAAG. The hOGG1 concentration is 0.1 U μL –1 and the hAAG concentration is 0.1 U μL –1 , and the APE1 concentration is 0.1 U μL –1 .

Article Snippet: Formation of the molecular beacons and the enzyme reactions Firstly, 6 μM hOGG1 substrate, 6 μM hAAG substrate and the mixture of 6 μM hOGG1 substrate and 6 μM hAAG substrate were incubated in 1× NEBuffer 2 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) at 95 °C for 5 min, respectively.

Techniques: Polyacrylamide Gel Electrophoresis, Labeling, Fluorescence, Concentration Assay

Journal: Chemical Science

Article Title: Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e

doi: 10.1039/c7sc04296e

Figure Lengend Snippet: Simultaneous detection of multiple DNA glycosylases by TIRF-based single-molecule imaging in the absence (A and E) and presence of hOGG1 (B and F), hAAG (C and G) and both hOGG1 and hAAG (D and H). The Cy3 fluorescence signals are shown in green, and the Cy5 fluorescence signals are shown in red. The Cy3-labeled molecular beacon (0.3 μM), the Cy5-labeled molecular beacon (0.3 μM), hOGG1 (0.1 U μL –1 ), hAAG (0.1 U μL –1 ) and APE1 (0.1 U μL –1 ) were used in this research. The scale bar is 5 μm.

Article Snippet: Formation of the molecular beacons and the enzyme reactions Firstly, 6 μM hOGG1 substrate, 6 μM hAAG substrate and the mixture of 6 μM hOGG1 substrate and 6 μM hAAG substrate were incubated in 1× NEBuffer 2 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) at 95 °C for 5 min, respectively.

Techniques: Imaging, Fluorescence, Labeling

Journal: Chemical Science

Article Title: Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e

doi: 10.1039/c7sc04296e

Figure Lengend Snippet: Measurement of the Cy3 counts and Cy5 counts in response to the reaction buffer (control), 0.1 g L –1 BSA, 0.1 U μL –1 UDG, 0.1 U μL –1 TDG, 0.1 U μL –1 hOGG1, 0.1 U μL –1 hAAG and 0.1 U μL –1 hOGG1 + 0.1 U μL –1 hAAG. The Cy3-labeled molecular beacon (0.3 μM), Cy5-labeled molecular beacon (0.3 μM) and APE1 (0.1 U μL –1 ) were used in this research. The error bars represent the standard deviations of the three experiments.

Article Snippet: Formation of the molecular beacons and the enzyme reactions Firstly, 6 μM hOGG1 substrate, 6 μM hAAG substrate and the mixture of 6 μM hOGG1 substrate and 6 μM hAAG substrate were incubated in 1× NEBuffer 2 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) at 95 °C for 5 min, respectively.

Techniques: Labeling

Journal: Chemical Science

Article Title: Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e

doi: 10.1039/c7sc04296e

Figure Lengend Snippet: (A) Measurement of the Cy3 counts generated by different concentrations of hOGG1. The inset shows the linear relationship between the Cy3 counts and the logarithm of the hOGG1 concentration. The Cy3-labeled molecular beacon (0.3 μM) and APE1 (0.1 U μL –1 ) were used in this research. (B) Measurement of the Cy5 counts generated by different concentrations of hAAG. The inset shows the linear relationship between the Cy5 counts and the logarithm of the hAAG concentration. The Cy5-labeled molecular beacon (0.3 μM) and APE1 (0.1 U μL –1 ) were used in this research. The error bars represent the standard deviations of the three experiments.

Article Snippet: Formation of the molecular beacons and the enzyme reactions Firstly, 6 μM hOGG1 substrate, 6 μM hAAG substrate and the mixture of 6 μM hOGG1 substrate and 6 μM hAAG substrate were incubated in 1× NEBuffer 2 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) at 95 °C for 5 min, respectively.

Techniques: Generated, Concentration Assay, Labeling

Journal: Chemical Science

Article Title: Programming in situ accelerated DNA walkers in diffusion-limited microenvironments accelerated DNA walkers in diffusion-limited microenvironments †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc05302b

doi: 10.1039/c8sc05302b

Figure Lengend Snippet: Schematic illustration of the comparison of in situ accelerated and routine post-assembly nanomotors in diffusion-limited microenvironments such as the cytoplasm. Nanosized modules (blue spheres) of size smaller than the cytoskeleton pore size (50–70 nm) may still diffuse (black polylines) slowly due to their non-specific interaction with immobile intracellular components. The endogenous proteins (gray shapes) are inert and can diffuse fast. ‘All’ highlighted spheres represent the integration of all modules into one system. In the post-assembly process, the motor can only be driven after the assembly of A and B modules. The proposed DNA walkers are powered by glycosylase and APE1. Crystal structures of chemically damaged (oG or AP site) DNA-bound human APE1 (PDB ID:; 1DEW ) and hOGG1 (PDB ID:; 1EBM ) were visualized and analyzed with PyMOL. The red circles indicate the locations of oG and AP sites in the crystal structures. The location of hAAG bound to I damage is not given.

Article Snippet: Human 8-oxoguanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAAG), human apurinic/apyrimidinic endonuclease 1 (APE1), and CutSmart (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(Ac)2 , 100 μg mL–1 BSA, pH 7.9) were purchased from New England Biolabs Inc. (Beverly, MA, U.S.A.).

Techniques: In Situ, Diffusion-based Assay

Journal: Nature Communications

Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression

doi: 10.1038/s41467-019-13394-w

Figure Lengend Snippet: Lack of functional Elongator hampers AAG and APE1 chromatin recruitment, and AAG-initiated repair. a–d ChIP-qPCR experiments comparing AAG occupancy at promoters and gene bodies of ALDH1A2 ( a ), CRMP1 ( b ), CDH23 ( c ), and YTHDC1 ( d ) in HEK293T WT and ELP1 −/− cells. e Immunoblot analysis of AAG levels in chromatin fraction of HEK293T WT and ELP1 −/− cells. Histone H3 served as control. f Quantification of experiments as the one depicted in e . g ChIP-qPCR experiments comparing APE1 occupancy at gene bodies of YTHDC1 , ALDH1A2 , CRMP1 , CDH23 , SYT9, and CDH4 in HEK293T WT and ELP1 −/− cells. Error bars represent the SEM calculated from at least three independent experiments. h Immunoprecipitation of AAG from HEK293T WT and ELP1 −/− whole cell extracts showing the interaction with RNA polymerase II phosphorylated at Serine 2 (S2P) of CTD repeat. i Measurement of AAG DNA glycosylase activity in HEK293T WT, AAG −/− and ELP1 −/− on hypoxanthine (Hx)-containing plasmid by FM-HCR assays. j AAG DNA glycosylase activity determined by FM-HCR assays on hypoxanthine (Hx)-containing plasmid in HEK293T WT cells complemented with GFP and AAG −/− cells complemented with GFP, GFP-AAG or GFP-Δ80 AAG. Error bars represent mean ± SEM ( n ≥ 3). Two-tailed Student’s t -test ( a – d , f , g ); one-way ANOVA ( i , j ), NS – non significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Source data are provided as Source Data file.

Article Snippet: DNA was incubated with either a combination of 10U of AAG (NEB, M0313) and 10U of APE1 (NEB, M0282), or only with 10U of APE1 for 1 h at 37 °C in 1X ThermoPol Reaction Buffer (NEB, B9004).

Techniques: Functional Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Activity Assay, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression

doi: 10.1038/s41467-019-13394-w

Figure Lengend Snippet: Model of AAG-initiated DNA repair coordinated with gene expression. Elongator complex associates with RNA pol II to promote transcription elongation, accumulating towards 3′end of regulated genes. AAG through its unstructured N-terminal region associates with ELP1 subunit of Elongator, thus forming complex with active transcription machinery. During active transcription chromatin is locally decondensed, which allows AAG to efficiently initiate BER by recognizing and removing aberrant bases. AAG-initiated BER likely temporarily inhibits RNA pol II progression, thus resulting in reduced expression of co-regulated genes. In the absence of Elongator, transcription of target genes is repressed, while AAG recruitment to chromatin and initiation of BER is impaired. For more details see text. Schematic representation was created with Biorender.com.

Article Snippet: DNA was incubated with either a combination of 10U of AAG (NEB, M0313) and 10U of APE1 (NEB, M0282), or only with 10U of APE1 for 1 h at 37 °C in 1X ThermoPol Reaction Buffer (NEB, B9004).

Techniques: Expressing

Journal: Nature Communications

Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression

doi: 10.1038/s41467-019-13394-w

Figure Lengend Snippet: Elongator, components of AAG-initiated BER and AAG substrates accumulate towards the 3′end of co-regulated genes. a AAG ChIP-qPCR assays in HEK293T WT cells comparing percentage of input at gene bodies of unaffected gene ( YTHDC1 ) and differentially expressed genes ( ALDH1A2, CRMP1, CDH23, SYT9 , and CDH4 ). b–e ChIP-qPCR assays showing relative AAG occupancy in AAG- and ELP1- dependent genes ALDH1A2 ( b ), CDH23 ( c ), CRMP1 ( d ), and unaffected gene YTHDC1 ( e ) in HEK293T WT cells. f–h ChIP-qPCR assays showing relative occupancy of HA-ELP1 in AAG- and ELP1- dependent ALDH1A2 ( f ), CDH23 ( g ), and CRMP1 ( h ) genes in HEK293T HA-ELP1cells. i–l ChIP-qPCR assays showing relative APE1 occupancy in negative control YTHDC1 gene ( i ), and AAG- and ELP1-dependent ALDH1A2 ( j ), CDH23 ( k ), CRMP1 ( l ) genes in HEK293T WT cells. m–p qPCR DNA damage assay showing differences in distribution pattern of aberrant AAG substrates in unaffected gene Y THDC1 ( m ) and genes regulated by AAG and ELP1: ALDH1A2 ( n ), CDH23 ( o ), CRMP1 ( p ) in HEK293T WT cells. Values are shown as relative occupancy: % input of specific gene region, relative to percentage input of promoter region. Error bars indicate mean ± SEM ( n ≥ 3). Two-tailed Student’s t -test in a ; one-way ANOVA in b – p ; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Source data are provided as Source Data file.

Article Snippet: DNA was incubated with either a combination of 10U of AAG (NEB, M0313) and 10U of APE1 (NEB, M0282), or only with 10U of APE1 for 1 h at 37 °C in 1X ThermoPol Reaction Buffer (NEB, B9004).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Mapping of DNA damage genome-wide at nucleotide resolution by circle damage sequencing (CD-seq)

doi: 10.1101/2020.06.28.176388

Figure Lengend Snippet: Genome-wide mapping of etheno-dA adducts using CD-seq. A. Etheno-dA adducts were induced in the THLE-2 genome by treatment with 40 µM of 4-HNE. To create double-strand breaks at etheno-dA (A with red symbol), the etheno-dA specific-DNA glycosylase hAAG and APE1/AP lyase coupled enzyme reactions were initially used to create nicked DNA. Then, S1 nuclease was used to generate a free 5’ phosphate and 3’ hydroxyl group at cleaved DNA ends, which are ligated and sequenced. B. The snapshot of the IGV viewer displays the divergent, paired reads of etheno-dA mapping by CD-seq. The distribution map shows 6.6 kb windows (chr10:93,940,000-93,946,591) of the hg19 human reference genome. Each paired-end read derived from a single DNA molecule was displayed as a single divergent pair (paired blue and red segments). 4-HNE; 4-HNE treated THLE-2 sample. NT; non-treated control THLE-2 sample showing almost no paired, divergent reads with single base gaps. C. The snapshot of the IGV viewer represents etheno-dA sites mapped by CD-seq at single-base resolution (chr17:8,302,652-8,302,754). Red segments represent reads mapped to the plus strand (forward) and blue segments represent reads mapped to the plus strand (reverse). Green arrows indicate single-base gaps matching etheno-dA bases in the reference sequence. D. Percentages of nucleotides positioned in the gap between divergent reads. ‘G + C’ and ‘A + T’ indicate the combined percentages of G and C or A and T in the gaps, respectively. E. Logo plot bit scores of DNA bases modified in 4-HNE-treated cells and in non-treated (NT) controls. This analysis considers both DNA strands. F. Logo plot bit scores of modified adenine bases in 4-HNE-treated cells and in non-treated (NT) controls. This analysis is specific for adenine bases in the gapped positions and considered the single strand-specific DNA sequence context.

Article Snippet: To generate double-strand breaks at etheno-dA adduct sites, the circularized DNA was initially treated with human alkyl adenine DNA glycosylase (hAAG, NEB) to cleave the N -glycosidic bond of etheno-dA bases.

Techniques: Genome Wide, Derivative Assay, Sequencing, Modification

Journal: bioRxiv

Article Title: Mapping of DNA damage genome-wide at nucleotide resolution by circle damage sequencing (CD-seq)

doi: 10.1101/2020.06.28.176388

Figure Lengend Snippet: Genome-wide mapping of CPD lesions and visualization at single nucleotide resolution. A. CPD adducts at dipyrimidine sequences were induced into the MEF genome by UVB irradiation with different doses (600 to 1500 J/m 2 ). To create double-strand breaks at CPDs (e.g., T=T; thymine dimer, red), CPD specific-DNA glycosylase (T4-PDG) and APE1 coupled enzyme reactions were initially used and followed by E . coli photolyase treatment to create ligatable, nicked DNA. To create double strand breaks, S1 nuclease was used to generate a free 5’ phosphate and 3’ hydroxyl group at cleaved DNA ends, which are ligated and sequenced. B. Snapshot of the IGV viewer displays the characteristic divergent paired reads of CPD mapping by CD-seq aligned to the mm10 mouse reference genome (chr6:113,533,595-113,533,704) at single-base resolution. Red segments represent reads mapped to the plus strand (forward) and blue segments represent reads mapped to the plus strand (reverse). Each paired-end read is derived from a single DNA circle and is displayed as a single divergent pair. Green arrows indicate single-base gaps matching pyrimidine bases on the reference sequence corresponding to the 5’ base of a pyrimidine dimer. C. Snapshot of the IGV viewer represents CPD sites mapped by CD-seq at an aligned region (chr6:114,103,889-114,103,928) at single-base resolution.

Article Snippet: To generate double-strand breaks at etheno-dA adduct sites, the circularized DNA was initially treated with human alkyl adenine DNA glycosylase (hAAG, NEB) to cleave the N -glycosidic bond of etheno-dA bases.

Techniques: Genome Wide, Irradiation, Derivative Assay, Sequencing