aag  (New England Biolabs)


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    Name:
    Human Alkyladenine Glycosylase hAAG
    Description:
    Human Alkyladenine Glycosylase hAAG 500 units
    Catalog Number:
    m0313s
    Price:
    76
    Size:
    500 units
    Category:
    DNA Glycosylases
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    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 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    aag - by Bioz Stars, 2020-04
    99/100 stars

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    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

    2) 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

    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

    3) 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

    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

    DNA Extraction:

    Article Title: Integrated genomic characterization of IDH1-mutant glioma malignant progression
    Article Snippet: For formalin-fixed, paraffin-embedded tissues, tumor DNA was extracted using the BiOstic FFPE Tissue DNA Isolation kit (MO BIO Laboratories, 12250-50). .. Extracted DNA was repaired using BioLabs PreCR Repair Mix (New England BioLabs, M0309S) with human alkyladenine DNA glycosylase (hAAG) (New England BioLabs, M0313S).

    SYBR Green Assay:

    Article Title: Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification
    Article Snippet: 4.1 Materials and Reagents Uracil-DNA glycosylase (UDG), human alkyladenine glycosylase (hAAG), uracil glycosylase inhibitor (UGI), T4 DNA ligase, exonuclease I (Exo I), exonuclease III (Exo III), endonuclease IV (Endo IV), and 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2 , 10 mM dithiothreitol) were purchased from New England Biolabs (Ipswich, MA, USA). .. 10× SYBR Green I was purchased from Zeesan (Xiamen, China).

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

    High Performance Liquid Chromatography:

    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
    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.). .. HPLC-purified oligonucleotides (Table S1 ) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and Takara Biotechnology Co. Ltd. (Dalian, China).

    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
    Article Snippet: Materials Human 8-oxoguanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAAG), human apurinic/apyrimidinic endonuclease 1 (APE1), 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris–HCl, 100 mM MgCl2 , 10 mM DTT, pH 7.9), 10× ThermoPol reaction buffer (200 mM Tris–HCl, 100 mM (NH4 )2 SO4 , 100 mM KCl, 20 mM MgSO4 , 1% Triton X-100, pH 8.8) and 10× NEBuffer 4 (500 mM potassium acetate, 200 mM Tris–acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9) were purchased from New England Biolabs Inc. (Beverly, MA, U.S.A.). .. All HPLC-purified oligonucleotides ( ) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

    Agarose Gel Electrophoresis:

    Article Title: The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa
    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. .. Samples were resolved on a 1.2% alkaline agarose gel (1.5 M NaOH, 50 mM EDTA).

    Synthesized:

    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
    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.). .. HPLC-purified oligonucleotides (Table S1 ) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and Takara Biotechnology Co. Ltd. (Dalian, China).

    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
    Article Snippet: Materials Human 8-oxoguanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAAG), human apurinic/apyrimidinic endonuclease 1 (APE1), 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris–HCl, 100 mM MgCl2 , 10 mM DTT, pH 7.9), 10× ThermoPol reaction buffer (200 mM Tris–HCl, 100 mM (NH4 )2 SO4 , 100 mM KCl, 20 mM MgSO4 , 1% Triton X-100, pH 8.8) and 10× NEBuffer 4 (500 mM potassium acetate, 200 mM Tris–acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9) were purchased from New England Biolabs Inc. (Beverly, MA, U.S.A.). .. All HPLC-purified oligonucleotides ( ) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

    Isolation:

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

    Protein Extraction:

    Article Title: Integrated genomic characterization of IDH1-mutant glioma malignant progression
    Article Snippet: Paragraph title: DNA, RNA and protein extraction ... Extracted DNA was repaired using BioLabs PreCR Repair Mix (New England BioLabs, M0309S) with human alkyladenine DNA glycosylase (hAAG) (New England BioLabs, M0313S).

    Labeling:

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

    Purification:

    Article Title: Integrated genomic characterization of IDH1-mutant glioma malignant progression
    Article Snippet: Extracted DNA was repaired using BioLabs PreCR Repair Mix (New England BioLabs, M0309S) with human alkyladenine DNA glycosylase (hAAG) (New England BioLabs, M0313S). .. Repaired DNA was subsequently purified using the QIAquick Nucleotide Removal kit (Qiagen, 28304).

    Electrophoresis:

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

    Concentration Assay:

    Article Title: The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa
    Article Snippet: BER assay Cells were grown for 11 hours in liquid VMM prior to addition of MMS to a final concentration of 0.035% MMS. .. 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.

    Incubation:

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

    Article Title: The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa
    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. .. Agarose gels were run in the cold room at 25V for 17hrs and then incubated in neutralization buffer (1.5 M NaCl, 1 M Tris-Cl pH 7.6) for 45 minutes before being stained with SYBR Gold (cat # S-11494, Life Technologies) for 40min and de-stained for 30 min before imaging.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Integrated genomic characterization of IDH1-mutant glioma malignant progression
    Article Snippet: For formalin-fixed, paraffin-embedded tissues, tumor DNA was extracted using the BiOstic FFPE Tissue DNA Isolation kit (MO BIO Laboratories, 12250-50). .. Extracted DNA was repaired using BioLabs PreCR Repair Mix (New England BioLabs, M0309S) with human alkyladenine DNA glycosylase (hAAG) (New England BioLabs, M0313S).

    Microscopy:

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

    Neutralization:

    Article Title: The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa
    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. .. Agarose gels were run in the cold room at 25V for 17hrs and then incubated in neutralization buffer (1.5 M NaCl, 1 M Tris-Cl pH 7.6) for 45 minutes before being stained with SYBR Gold (cat # S-11494, Life Technologies) for 40min and de-stained for 30 min before imaging.

    Staining:

    Article Title: The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa
    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. .. Agarose gels were run in the cold room at 25V for 17hrs and then incubated in neutralization buffer (1.5 M NaCl, 1 M Tris-Cl pH 7.6) for 45 minutes before being stained with SYBR Gold (cat # S-11494, Life Technologies) for 40min and de-stained for 30 min before imaging.

    Lysis:

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

    other:

    Article Title: A single quantum dot-based nanosensor with multilayer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase single quantum dot-based nanosensor with multilayer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc02137j
    Article Snippet: Human alkyladenine DNA glycosylase (hAAG), human apurinic/apyrimidinic endonuclease 1 (APE1), Klenow fragment (3′ → 5′ exo– ), human 8-oxoguanine-DNA glycosylase 1 (hOGG1), uracil DNA glycosylase (UDG), T4 polynucleotide kinase (PNK), 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris–HCl, 100 mM MgCl2 , 10 mM DTT, pH 7.9), and 10× NEBuffer 4 (500 mM potassium acetate, 200 mM Tris–acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9) were purchased from New England Biolabs (Ipswich, MA, USA).

    Imaging:

    Article Title: The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa
    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. .. Agarose gels were run in the cold room at 25V for 17hrs and then incubated in neutralization buffer (1.5 M NaCl, 1 M Tris-Cl pH 7.6) for 45 minutes before being stained with SYBR Gold (cat # S-11494, Life Technologies) for 40min and de-stained for 30 min before imaging.

    Software:

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    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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    New England Biolabs human alkyladenine dna glycosylase
    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 <t>DNA</t> walkers are powered by <t>glycosylase</t> 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.
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    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

    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.

    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

    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.

    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

    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.

    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

    Schematic illustration of the multiple DNA glycosylase assay using the DNA glycosylase-mediated cleavage of molecular beacons and single-molecule detection.

    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: Schematic illustration of the multiple DNA glycosylase assay using the DNA glycosylase-mediated cleavage of molecular beacons and single-molecule detection.

    Article Snippet: Materials Human 8-oxoguanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAAG), human apurinic/apyrimidinic endonuclease 1 (APE1), 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris–HCl, 100 mM MgCl2 , 10 mM DTT, pH 7.9), 10× ThermoPol reaction buffer (200 mM Tris–HCl, 100 mM (NH4 )2 SO4 , 100 mM KCl, 20 mM MgSO4 , 1% Triton X-100, pH 8.8) and 10× NEBuffer 4 (500 mM potassium acetate, 200 mM Tris–acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9) were purchased from New England Biolabs Inc. (Beverly, MA, U.S.A.).

    Techniques:

    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