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Name:

Human Alkyladenine Glycosylase hAAG

Description:

Human Alkyladenine Glycosylase hAAG 500 units

Catalog Number:

m0313s

Price:

Size:

500 units

Category:

DNA Glycosylases

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Structured Review

New England Biolabs aag

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

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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

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:

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:

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 ).