aag  (New England Biolabs)


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    Name:
    Human Alkyladenine Glycosylase hAAG
    Description:
    Human Alkyladenine Glycosylase hAAG 500 units
    Catalog Number:
    m0313s
    Price:
    75
    Size:
    500 units
    Category:
    DNA Glycosylases
    Buy from Supplier


    Structured Review

    New England Biolabs aag
    Human Alkyladenine Glycosylase hAAG
    Human Alkyladenine Glycosylase hAAG 500 units
    https://www.bioz.com/result/aag/product/New England Biolabs
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    aag - by Bioz Stars, 2020-01
    78/100 stars

    Images

    1) Product Images from "Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13394-w

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing

    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.
    Figure Legend 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.

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

    Related Articles

    Electrophoresis:

    Article Title:
    Article Snippet: Next, detergent was removed by washing and the slides were incubated for 1 h at 37 °C in the presence of 2U of AAG (NEB, M0313) in reaction buffer (2.5 mM HEPES-KOH pH 7.4, 100 mM KCl, 25 mM EDTA, 2 mM BSA). .. The DNA was subjected to alkaline electrophoresis (1 mM EDTA, 200 mM NaOH, pH > 13) and labeled with SYBR® Green (Sigma Aldrich).

    Microscopy:

    Article Title:
    Article Snippet: Next, detergent was removed by washing and the slides were incubated for 1 h at 37 °C in the presence of 2U of AAG (NEB, M0313) in reaction buffer (2.5 mM HEPES-KOH pH 7.4, 100 mM KCl, 25 mM EDTA, 2 mM BSA). .. Slides were imaged on a Zeiss Axiovert 200 M epifluorescent microscope using a ×20 objective and the percentage of the DNA in the tail was evaluated for each data point with Comet IV, v4.2 (Perceptive Instruments, Suffolk, U.K.).

    SYBR Green Assay:

    Article Title:
    Article Snippet: Next, detergent was removed by washing and the slides were incubated for 1 h at 37 °C in the presence of 2U of AAG (NEB, M0313) in reaction buffer (2.5 mM HEPES-KOH pH 7.4, 100 mM KCl, 25 mM EDTA, 2 mM BSA). .. The DNA was subjected to alkaline electrophoresis (1 mM EDTA, 200 mM NaOH, pH > 13) and labeled with SYBR® Green (Sigma Aldrich).

    Incubation:

    Article Title:
    Article Snippet: .. Next, detergent was removed by washing and the slides were incubated for 1 h at 37 °C in the presence of 2U of AAG (NEB, M0313) in reaction buffer (2.5 mM HEPES-KOH pH 7.4, 100 mM KCl, 25 mM EDTA, 2 mM BSA). .. The DNA was subjected to alkaline electrophoresis (1 mM EDTA, 200 mM NaOH, pH > 13) and labeled with SYBR® Green (Sigma Aldrich).

    Article Title:
    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). .. Next, level of DNA damage was assessed by qPCR (StepOne Software version v2.3) using primers targeting regions corresponding to promoter, middle and end of the gene of interest (Supplementary Table ).

    Labeling:

    Article Title:
    Article Snippet: Next, detergent was removed by washing and the slides were incubated for 1 h at 37 °C in the presence of 2U of AAG (NEB, M0313) in reaction buffer (2.5 mM HEPES-KOH pH 7.4, 100 mM KCl, 25 mM EDTA, 2 mM BSA). .. The DNA was subjected to alkaline electrophoresis (1 mM EDTA, 200 mM NaOH, pH > 13) and labeled with SYBR® Green (Sigma Aldrich).

    Lysis:

    Article Title:
    Article Snippet: Next cells were collected and mixed with 1% of low melting point agarose, spread onto glass slides (Trevigen) and lysed overnight in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton, 10% DMSO, 1% sodium lauryl sarcosinate). .. Next, detergent was removed by washing and the slides were incubated for 1 h at 37 °C in the presence of 2U of AAG (NEB, M0313) in reaction buffer (2.5 mM HEPES-KOH pH 7.4, 100 mM KCl, 25 mM EDTA, 2 mM BSA).

    Real-time Polymerase Chain Reaction:

    Article Title:
    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). .. Next, level of DNA damage was assessed by qPCR (StepOne Software version v2.3) using primers targeting regions corresponding to promoter, middle and end of the gene of interest (Supplementary Table ).

    Software:

    Article Title:
    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). .. Next, level of DNA damage was assessed by qPCR (StepOne Software version v2.3) using primers targeting regions corresponding to promoter, middle and end of the gene of interest (Supplementary Table ).

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  • 85
    New England Biolabs human alkyladenine glycosylase
    Δmus-30 interacts genetically with Δmag-1 and Δmei-3 . (A) Serial dilutions of conidia (10 4 −10 1 ) were spot tested on minimal medium (VMM) with or without the indicated concentrations of MMS for the indicated strains. (B) The average number of colonies for each genotype is shown for the indicated concentrations of MMS. For each concentration, % survival is shown relative to no MMS control. At least two isolates of each genotype were analyzed. Error bars show the standard deviation. (C) Repair of MMS-induced damage is shown in wildtype, Δmus-30 , and Δmag-1 cells, as indicated. Genomic DNA was isolated from cells before, during, and after MMS exposure, as indicated. DNA was treated with Human <t>Alkyladenine</t> DNA <t>Glycosylase</t> <t>(hAAG),</t> apurinic/apyrimidinic endonuclease (APE), or both to induce ssDNA breaks at methylated bases or abasic sites. The size of ssDNA was visualized at each time point by alkaline electrophoresis. A low molecular weight smear indicates the presence of unrepaired DNA after MMS treatment. (D) Images of race tubes containing minimal medium show the relative growth rates of the indicated strains. (E) The linear growth rate is plotted for multiple isolates of each genotype shown in D. (F) Serial dilutions of conidia (10 4 −10 1 ) were spotted on minimal medium (VMM) with or without the indicated concentrations of MMS for wildtype and the indicated single mutants. For Δmei-3 ; Δmus-30 strains, a dilution series from 10 5 −10 2 was used due to poor spore viability (asterisk).
    Human Alkyladenine Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human alkyladenine glycosylase/product/New England Biolabs
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    human alkyladenine glycosylase - by Bioz Stars, 2020-01
    85/100 stars
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    Δmus-30 interacts genetically with Δmag-1 and Δmei-3 . (A) Serial dilutions of conidia (10 4 −10 1 ) were spot tested on minimal medium (VMM) with or without the indicated concentrations of MMS for the indicated strains. (B) The average number of colonies for each genotype is shown for the indicated concentrations of MMS. For each concentration, % survival is shown relative to no MMS control. At least two isolates of each genotype were analyzed. Error bars show the standard deviation. (C) Repair of MMS-induced damage is shown in wildtype, Δmus-30 , and Δmag-1 cells, as indicated. Genomic DNA was isolated from cells before, during, and after MMS exposure, as indicated. DNA was treated with Human Alkyladenine DNA Glycosylase (hAAG), apurinic/apyrimidinic endonuclease (APE), or both to induce ssDNA breaks at methylated bases or abasic sites. The size of ssDNA was visualized at each time point by alkaline electrophoresis. A low molecular weight smear indicates the presence of unrepaired DNA after MMS treatment. (D) Images of race tubes containing minimal medium show the relative growth rates of the indicated strains. (E) The linear growth rate is plotted for multiple isolates of each genotype shown in D. (F) Serial dilutions of conidia (10 4 −10 1 ) were spotted on minimal medium (VMM) with or without the indicated concentrations of MMS for wildtype and the indicated single mutants. For Δmei-3 ; Δmus-30 strains, a dilution series from 10 5 −10 2 was used due to poor spore viability (asterisk).

    Journal: PLoS Genetics

    Article Title: The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa

    doi: 10.1371/journal.pgen.1005790

    Figure Lengend Snippet: Δmus-30 interacts genetically with Δmag-1 and Δmei-3 . (A) Serial dilutions of conidia (10 4 −10 1 ) were spot tested on minimal medium (VMM) with or without the indicated concentrations of MMS for the indicated strains. (B) The average number of colonies for each genotype is shown for the indicated concentrations of MMS. For each concentration, % survival is shown relative to no MMS control. At least two isolates of each genotype were analyzed. Error bars show the standard deviation. (C) Repair of MMS-induced damage is shown in wildtype, Δmus-30 , and Δmag-1 cells, as indicated. Genomic DNA was isolated from cells before, during, and after MMS exposure, as indicated. DNA was treated with Human Alkyladenine DNA Glycosylase (hAAG), apurinic/apyrimidinic endonuclease (APE), or both to induce ssDNA breaks at methylated bases or abasic sites. The size of ssDNA was visualized at each time point by alkaline electrophoresis. A low molecular weight smear indicates the presence of unrepaired DNA after MMS treatment. (D) Images of race tubes containing minimal medium show the relative growth rates of the indicated strains. (E) The linear growth rate is plotted for multiple isolates of each genotype shown in D. (F) Serial dilutions of conidia (10 4 −10 1 ) were spotted on minimal medium (VMM) with or without the indicated concentrations of MMS for wildtype and the indicated single mutants. For Δmei-3 ; Δmus-30 strains, a dilution series from 10 5 −10 2 was used due to poor spore viability (asterisk).

    Article Snippet: Genomic DNA was isolated and 300 ng was digested by AP endonuclease (cat # M0282S, New England Biolabs), human alkyladenine DNA Glycosylase (cat # M0313S, New England Biolabs), or both enzymes for 1hr and 15min at 37C.

    Techniques: Concentration Assay, Standard Deviation, Isolation, Methylation, Electrophoresis, Molecular Weight

    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.

    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