a3h catalyzed deaminations  (New England Biolabs)


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    New England Biolabs a3h catalyzed deaminations
    β2-β2 strand amino acids mediate <t>A3H</t> dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.
    A3h Catalyzed Deaminations, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a3h catalyzed deaminations/product/New England Biolabs
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    a3h catalyzed deaminations - by Bioz Stars, 2020-08
    88/100 stars

    Related Products / Commonly Used Together

    uracil dna glycosylase
    ssdna

    Images

    1) Product Images from "Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability *"

    Article Title: Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.666065

    β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.
    Figure Legend Snippet: β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.

    Techniques Used: Size-exclusion Chromatography, Concentration Assay, Sequencing, Construct, SDS Page

    2) Product Images from "RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids"

    Article Title: RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2018.11.006

    A3H alters physical properties of ssDNA upon binding. (A) The binding of A3H WT (blue) or Y112A/Y113A (red) greatly increases the persistence length of ssDNA (green). Symbols are experimental data and lines are fits of the wormlike chain (dsDNA) or FJC model. (B) The extended length of ssDNA increases while held at a constant tension of 30 pN while incubated with 1 μM A3H as shown by two sample curves. Once the free protein is removed from the sample, some bound A3H dissociates resulting in a partial reversal of the extension change. (C) The measured persistence length of ssDNA is greatly increased both after incubating with 1 μM A3H for 5 min (+) and after allowing bound protein to dissociate for 5 min in the absence of free protein (−). (D) ssDNA forms hairpins and loops in the absence of applied force, resulting in a shorter apparent length when stretched to high force (solid line) versus when relaxed to low force (open symbols). For bare ssDNA (green), hairpins are completely removed above ~15 pN of applied force, but bound A3H WT (blue) or Y112A/Y113A (red) stabilizes these structures requiring higher forces and longer times to break apart. Both the ability to locally straighten ssDNA and stabilize loop formation is enhanced for A3H Y112A/Y113A as compared to A3H WT.
    Figure Legend Snippet: A3H alters physical properties of ssDNA upon binding. (A) The binding of A3H WT (blue) or Y112A/Y113A (red) greatly increases the persistence length of ssDNA (green). Symbols are experimental data and lines are fits of the wormlike chain (dsDNA) or FJC model. (B) The extended length of ssDNA increases while held at a constant tension of 30 pN while incubated with 1 μM A3H as shown by two sample curves. Once the free protein is removed from the sample, some bound A3H dissociates resulting in a partial reversal of the extension change. (C) The measured persistence length of ssDNA is greatly increased both after incubating with 1 μM A3H for 5 min (+) and after allowing bound protein to dissociate for 5 min in the absence of free protein (−). (D) ssDNA forms hairpins and loops in the absence of applied force, resulting in a shorter apparent length when stretched to high force (solid line) versus when relaxed to low force (open symbols). For bare ssDNA (green), hairpins are completely removed above ~15 pN of applied force, but bound A3H WT (blue) or Y112A/Y113A (red) stabilizes these structures requiring higher forces and longer times to break apart. Both the ability to locally straighten ssDNA and stabilize loop formation is enhanced for A3H Y112A/Y113A as compared to A3H WT.

    Techniques Used: Binding Assay, Incubation

    Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.
    Figure Legend Snippet: Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.

    Techniques Used: Activity Assay

    3) Product Images from "Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer"

    Article Title: Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

    Journal: bioRxiv

    doi: 10.1101/2020.02.28.970509

    A3H Hap I mutant dimerization, oligomerization, and ssDNA binding. a) A3H dimerization is mediated by RNA. a) Purified A3H was denatured in formamide buffer and samples were resolved by urea denaturing PAGE. The gel was stained with SYBR Gold to detect nucleic acids. The A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II protect an approximately 12-15 nt RNA. Representative image shown from three independent experiments. b) Standard curve and c-d) SEC profile for A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II demonstrating similar elution profiles that are composed primarily of a dimer peak (44 kDa, 17 mL elution volume). (e-h) The apparent K d of A3H enzymes from a 118 nt ssDNA was analyzed by steady-state rotational anisotropy. The e) A3H Hap I K117E (3005 ± 1768 nM) and f) A3H Hap I K117E/K121E (1239 ± 88 nM) bind the ssDNA with different affinities than g) A3H Hap VII (522 ± 4 nM) and h) A3H Hap II (492 ± 28 nM). The apparent K d was calculated by determining the best fit by least squares analysis which was a hyperbolic fit for e-f and a sigmoidal fit for g-h . Error bars represent the S.D. from three independent experiments.
    Figure Legend Snippet: A3H Hap I mutant dimerization, oligomerization, and ssDNA binding. a) A3H dimerization is mediated by RNA. a) Purified A3H was denatured in formamide buffer and samples were resolved by urea denaturing PAGE. The gel was stained with SYBR Gold to detect nucleic acids. The A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II protect an approximately 12-15 nt RNA. Representative image shown from three independent experiments. b) Standard curve and c-d) SEC profile for A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II demonstrating similar elution profiles that are composed primarily of a dimer peak (44 kDa, 17 mL elution volume). (e-h) The apparent K d of A3H enzymes from a 118 nt ssDNA was analyzed by steady-state rotational anisotropy. The e) A3H Hap I K117E (3005 ± 1768 nM) and f) A3H Hap I K117E/K121E (1239 ± 88 nM) bind the ssDNA with different affinities than g) A3H Hap VII (522 ± 4 nM) and h) A3H Hap II (492 ± 28 nM). The apparent K d was calculated by determining the best fit by least squares analysis which was a hyperbolic fit for e-f and a sigmoidal fit for g-h . Error bars represent the S.D. from three independent experiments.

    Techniques Used: Mutagenesis, Binding Assay, Purification, Polyacrylamide Gel Electrophoresis, Staining

    Expression and mutagenic activity of A3H Hap I. a) Transient expression of HA-tagged A3H Hap I expression constructs in 293T cells were detected by immunoblotting. The α-tubulin served as the loading control. The endogenous A3B and A3H Hap I mRNA levels were detected in b) MRC-5 and c) NCI-H1563 cells relative to TBP mRNA. The A3H Hap I mRNA levels were low, so doxycycline inducible A3H Hap I-Flag cell lines were produced (Figure SF6). (d-g) A3H Hap I induces high amounts of γH2AX foci in NCI-H1563 cells with endogenous A3B expression. The γH2AX foci were detected in induced and uninduced cells by immunofluorescence microscopy in d) MRC-5 and e) NCI-H1563 cells. Results were quantified and plotted as a histogram for f) MRC-5 and g) NCI-H1563 cells.
    Figure Legend Snippet: Expression and mutagenic activity of A3H Hap I. a) Transient expression of HA-tagged A3H Hap I expression constructs in 293T cells were detected by immunoblotting. The α-tubulin served as the loading control. The endogenous A3B and A3H Hap I mRNA levels were detected in b) MRC-5 and c) NCI-H1563 cells relative to TBP mRNA. The A3H Hap I mRNA levels were low, so doxycycline inducible A3H Hap I-Flag cell lines were produced (Figure SF6). (d-g) A3H Hap I induces high amounts of γH2AX foci in NCI-H1563 cells with endogenous A3B expression. The γH2AX foci were detected in induced and uninduced cells by immunofluorescence microscopy in d) MRC-5 and e) NCI-H1563 cells. Results were quantified and plotted as a histogram for f) MRC-5 and g) NCI-H1563 cells.

    Techniques Used: Expressing, Activity Assay, Construct, Produced, Immunofluorescence, Microscopy

    Deamination activity of A3H WT and mutants. a) Time course of deamination of A3H Hap II, A3H Hap VII, and A3H Hap I WT and mutants. Deamination was tested on a 118 nt ssDNA (100 nM) and gels analyzed for each plot are shown in Figure SF5. b) Processivity of A3H Hap I K117E and K117E/K121E. The processivity factor (P.F.) measures the likelihood of a processive deamination over a nonprocessive deamination and is described further in the Methods. Both K117E and K117E/K121E are approximately 6-fold more likely to undergo processive deamination than a nonprocessive deamination. The standard deviation for three independent experiments is shown as ( a ) error bars or ( b ) below the gel.
    Figure Legend Snippet: Deamination activity of A3H WT and mutants. a) Time course of deamination of A3H Hap II, A3H Hap VII, and A3H Hap I WT and mutants. Deamination was tested on a 118 nt ssDNA (100 nM) and gels analyzed for each plot are shown in Figure SF5. b) Processivity of A3H Hap I K117E and K117E/K121E. The processivity factor (P.F.) measures the likelihood of a processive deamination over a nonprocessive deamination and is described further in the Methods. Both K117E and K117E/K121E are approximately 6-fold more likely to undergo processive deamination than a nonprocessive deamination. The standard deviation for three independent experiments is shown as ( a ) error bars or ( b ) below the gel.

    Techniques Used: Activity Assay, Standard Deviation

    4) Product Images from "RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids"

    Article Title: RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2018.11.006

    A3H alters physical properties of ssDNA upon binding. (A) The binding of A3H WT (blue) or Y112A/Y113A (red) greatly increases the persistence length of ssDNA (green). Symbols are experimental data and lines are fits of the wormlike chain (dsDNA) or FJC model. (B) The extended length of ssDNA increases while held at a constant tension of 30 pN while incubated with 1 μM A3H as shown by two sample curves. Once the free protein is removed from the sample, some bound A3H dissociates resulting in a partial reversal of the extension change. (C) The measured persistence length of ssDNA is greatly increased both after incubating with 1 μM A3H for 5 min (+) and after allowing bound protein to dissociate for 5 min in the absence of free protein (−). (D) ssDNA forms hairpins and loops in the absence of applied force, resulting in a shorter apparent length when stretched to high force (solid line) versus when relaxed to low force (open symbols). For bare ssDNA (green), hairpins are completely removed above ~15 pN of applied force, but bound A3H WT (blue) or Y112A/Y113A (red) stabilizes these structures requiring higher forces and longer times to break apart. Both the ability to locally straighten ssDNA and stabilize loop formation is enhanced for A3H Y112A/Y113A as compared to A3H WT.
    Figure Legend Snippet: A3H alters physical properties of ssDNA upon binding. (A) The binding of A3H WT (blue) or Y112A/Y113A (red) greatly increases the persistence length of ssDNA (green). Symbols are experimental data and lines are fits of the wormlike chain (dsDNA) or FJC model. (B) The extended length of ssDNA increases while held at a constant tension of 30 pN while incubated with 1 μM A3H as shown by two sample curves. Once the free protein is removed from the sample, some bound A3H dissociates resulting in a partial reversal of the extension change. (C) The measured persistence length of ssDNA is greatly increased both after incubating with 1 μM A3H for 5 min (+) and after allowing bound protein to dissociate for 5 min in the absence of free protein (−). (D) ssDNA forms hairpins and loops in the absence of applied force, resulting in a shorter apparent length when stretched to high force (solid line) versus when relaxed to low force (open symbols). For bare ssDNA (green), hairpins are completely removed above ~15 pN of applied force, but bound A3H WT (blue) or Y112A/Y113A (red) stabilizes these structures requiring higher forces and longer times to break apart. Both the ability to locally straighten ssDNA and stabilize loop formation is enhanced for A3H Y112A/Y113A as compared to A3H WT.

    Techniques Used: Binding Assay, Incubation

    Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.
    Figure Legend Snippet: Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.

    Techniques Used: Activity Assay

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    New England Biolabs a3h catalyzed deaminations
    β2-β2 strand amino acids mediate <t>A3H</t> dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.
    A3h Catalyzed Deaminations, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a3h catalyzed deaminations/product/New England Biolabs
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    a3h catalyzed deaminations - by Bioz Stars, 2020-08
    88/100 stars
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    β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.

    Journal: The Journal of Biological Chemistry

    Article Title: Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability *

    doi: 10.1074/jbc.M115.666065

    Figure Lengend Snippet: β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.

    Article Snippet: A3H-catalyzed deaminations were detected by treating the ssDNA with uracil DNA glycosylase (New England Biolabs) and heating under alkaline conditions before resolving the fluorescein-labeled ssDNA on 10, 16, or 20% (v/v) denaturing polyacrylamide gels, depending on the sizes of the ssDNA fragments.

    Techniques: Size-exclusion Chromatography, Concentration Assay, Sequencing, Construct, SDS Page

    A3H alters physical properties of ssDNA upon binding. (A) The binding of A3H WT (blue) or Y112A/Y113A (red) greatly increases the persistence length of ssDNA (green). Symbols are experimental data and lines are fits of the wormlike chain (dsDNA) or FJC model. (B) The extended length of ssDNA increases while held at a constant tension of 30 pN while incubated with 1 μM A3H as shown by two sample curves. Once the free protein is removed from the sample, some bound A3H dissociates resulting in a partial reversal of the extension change. (C) The measured persistence length of ssDNA is greatly increased both after incubating with 1 μM A3H for 5 min (+) and after allowing bound protein to dissociate for 5 min in the absence of free protein (−). (D) ssDNA forms hairpins and loops in the absence of applied force, resulting in a shorter apparent length when stretched to high force (solid line) versus when relaxed to low force (open symbols). For bare ssDNA (green), hairpins are completely removed above ~15 pN of applied force, but bound A3H WT (blue) or Y112A/Y113A (red) stabilizes these structures requiring higher forces and longer times to break apart. Both the ability to locally straighten ssDNA and stabilize loop formation is enhanced for A3H Y112A/Y113A as compared to A3H WT.

    Journal: Journal of molecular biology

    Article Title: RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids

    doi: 10.1016/j.jmb.2018.11.006

    Figure Lengend Snippet: A3H alters physical properties of ssDNA upon binding. (A) The binding of A3H WT (blue) or Y112A/Y113A (red) greatly increases the persistence length of ssDNA (green). Symbols are experimental data and lines are fits of the wormlike chain (dsDNA) or FJC model. (B) The extended length of ssDNA increases while held at a constant tension of 30 pN while incubated with 1 μM A3H as shown by two sample curves. Once the free protein is removed from the sample, some bound A3H dissociates resulting in a partial reversal of the extension change. (C) The measured persistence length of ssDNA is greatly increased both after incubating with 1 μM A3H for 5 min (+) and after allowing bound protein to dissociate for 5 min in the absence of free protein (−). (D) ssDNA forms hairpins and loops in the absence of applied force, resulting in a shorter apparent length when stretched to high force (solid line) versus when relaxed to low force (open symbols). For bare ssDNA (green), hairpins are completely removed above ~15 pN of applied force, but bound A3H WT (blue) or Y112A/Y113A (red) stabilizes these structures requiring higher forces and longer times to break apart. Both the ability to locally straighten ssDNA and stabilize loop formation is enhanced for A3H Y112A/Y113A as compared to A3H WT.

    Article Snippet: A3H catalyzed deaminations were detected by treating the ssDNA with Uracil DNA Glycosylase (New England Biolabs) and heating under alkaline conditions before resolving the Fluorescein dT-labeled ssDNA on a 16% (v/v) denaturing polyacrylamide gel.

    Techniques: Binding Assay, Incubation

    Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.

    Journal: Journal of molecular biology

    Article Title: RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids

    doi: 10.1016/j.jmb.2018.11.006

    Figure Lengend Snippet: Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.

    Article Snippet: A3H catalyzed deaminations were detected by treating the ssDNA with Uracil DNA Glycosylase (New England Biolabs) and heating under alkaline conditions before resolving the Fluorescein dT-labeled ssDNA on a 16% (v/v) denaturing polyacrylamide gel.

    Techniques: Activity Assay

    A3H Hap I mutant dimerization, oligomerization, and ssDNA binding. a) A3H dimerization is mediated by RNA. a) Purified A3H was denatured in formamide buffer and samples were resolved by urea denaturing PAGE. The gel was stained with SYBR Gold to detect nucleic acids. The A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II protect an approximately 12-15 nt RNA. Representative image shown from three independent experiments. b) Standard curve and c-d) SEC profile for A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II demonstrating similar elution profiles that are composed primarily of a dimer peak (44 kDa, 17 mL elution volume). (e-h) The apparent K d of A3H enzymes from a 118 nt ssDNA was analyzed by steady-state rotational anisotropy. The e) A3H Hap I K117E (3005 ± 1768 nM) and f) A3H Hap I K117E/K121E (1239 ± 88 nM) bind the ssDNA with different affinities than g) A3H Hap VII (522 ± 4 nM) and h) A3H Hap II (492 ± 28 nM). The apparent K d was calculated by determining the best fit by least squares analysis which was a hyperbolic fit for e-f and a sigmoidal fit for g-h . Error bars represent the S.D. from three independent experiments.

    Journal: bioRxiv

    Article Title: Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

    doi: 10.1101/2020.02.28.970509

    Figure Lengend Snippet: A3H Hap I mutant dimerization, oligomerization, and ssDNA binding. a) A3H dimerization is mediated by RNA. a) Purified A3H was denatured in formamide buffer and samples were resolved by urea denaturing PAGE. The gel was stained with SYBR Gold to detect nucleic acids. The A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II protect an approximately 12-15 nt RNA. Representative image shown from three independent experiments. b) Standard curve and c-d) SEC profile for A3H Hap I K117E, A3H Hap I K117E/K121E, A3H Hap VII, and A3H Hap II demonstrating similar elution profiles that are composed primarily of a dimer peak (44 kDa, 17 mL elution volume). (e-h) The apparent K d of A3H enzymes from a 118 nt ssDNA was analyzed by steady-state rotational anisotropy. The e) A3H Hap I K117E (3005 ± 1768 nM) and f) A3H Hap I K117E/K121E (1239 ± 88 nM) bind the ssDNA with different affinities than g) A3H Hap VII (522 ± 4 nM) and h) A3H Hap II (492 ± 28 nM). The apparent K d was calculated by determining the best fit by least squares analysis which was a hyperbolic fit for e-f and a sigmoidal fit for g-h . Error bars represent the S.D. from three independent experiments.

    Article Snippet: A3H catalyzed deaminations were detected by treating the ssDNA with Uracil DNA Glycosylase (New England Biolabs) and heating under alkaline conditions before resolving the Fluorescein dT-labeled ssDNA on a 10% (v/v) denaturing polyacrylamide gel.

    Techniques: Mutagenesis, Binding Assay, Purification, Polyacrylamide Gel Electrophoresis, Staining

    Expression and mutagenic activity of A3H Hap I. a) Transient expression of HA-tagged A3H Hap I expression constructs in 293T cells were detected by immunoblotting. The α-tubulin served as the loading control. The endogenous A3B and A3H Hap I mRNA levels were detected in b) MRC-5 and c) NCI-H1563 cells relative to TBP mRNA. The A3H Hap I mRNA levels were low, so doxycycline inducible A3H Hap I-Flag cell lines were produced (Figure SF6). (d-g) A3H Hap I induces high amounts of γH2AX foci in NCI-H1563 cells with endogenous A3B expression. The γH2AX foci were detected in induced and uninduced cells by immunofluorescence microscopy in d) MRC-5 and e) NCI-H1563 cells. Results were quantified and plotted as a histogram for f) MRC-5 and g) NCI-H1563 cells.

    Journal: bioRxiv

    Article Title: Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

    doi: 10.1101/2020.02.28.970509

    Figure Lengend Snippet: Expression and mutagenic activity of A3H Hap I. a) Transient expression of HA-tagged A3H Hap I expression constructs in 293T cells were detected by immunoblotting. The α-tubulin served as the loading control. The endogenous A3B and A3H Hap I mRNA levels were detected in b) MRC-5 and c) NCI-H1563 cells relative to TBP mRNA. The A3H Hap I mRNA levels were low, so doxycycline inducible A3H Hap I-Flag cell lines were produced (Figure SF6). (d-g) A3H Hap I induces high amounts of γH2AX foci in NCI-H1563 cells with endogenous A3B expression. The γH2AX foci were detected in induced and uninduced cells by immunofluorescence microscopy in d) MRC-5 and e) NCI-H1563 cells. Results were quantified and plotted as a histogram for f) MRC-5 and g) NCI-H1563 cells.

    Article Snippet: A3H catalyzed deaminations were detected by treating the ssDNA with Uracil DNA Glycosylase (New England Biolabs) and heating under alkaline conditions before resolving the Fluorescein dT-labeled ssDNA on a 10% (v/v) denaturing polyacrylamide gel.

    Techniques: Expressing, Activity Assay, Construct, Produced, Immunofluorescence, Microscopy

    Deamination activity of A3H WT and mutants. a) Time course of deamination of A3H Hap II, A3H Hap VII, and A3H Hap I WT and mutants. Deamination was tested on a 118 nt ssDNA (100 nM) and gels analyzed for each plot are shown in Figure SF5. b) Processivity of A3H Hap I K117E and K117E/K121E. The processivity factor (P.F.) measures the likelihood of a processive deamination over a nonprocessive deamination and is described further in the Methods. Both K117E and K117E/K121E are approximately 6-fold more likely to undergo processive deamination than a nonprocessive deamination. The standard deviation for three independent experiments is shown as ( a ) error bars or ( b ) below the gel.

    Journal: bioRxiv

    Article Title: Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

    doi: 10.1101/2020.02.28.970509

    Figure Lengend Snippet: Deamination activity of A3H WT and mutants. a) Time course of deamination of A3H Hap II, A3H Hap VII, and A3H Hap I WT and mutants. Deamination was tested on a 118 nt ssDNA (100 nM) and gels analyzed for each plot are shown in Figure SF5. b) Processivity of A3H Hap I K117E and K117E/K121E. The processivity factor (P.F.) measures the likelihood of a processive deamination over a nonprocessive deamination and is described further in the Methods. Both K117E and K117E/K121E are approximately 6-fold more likely to undergo processive deamination than a nonprocessive deamination. The standard deviation for three independent experiments is shown as ( a ) error bars or ( b ) below the gel.

    Article Snippet: A3H catalyzed deaminations were detected by treating the ssDNA with Uracil DNA Glycosylase (New England Biolabs) and heating under alkaline conditions before resolving the Fluorescein dT-labeled ssDNA on a 10% (v/v) denaturing polyacrylamide gel.

    Techniques: Activity Assay, Standard Deviation