hsmug1  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: Indication of hSMUG1 incision at uracil in DNA. ( A ) DNA substrate and conventional base excision assay. ( B, C ) Protein dependence of U-DNA incision (red) and uracil excision (blue). hSMUG1 was incubated with U-DNA (substrate 1, 0.5 pmol) in 20 mM Tris–HCl, pH 8.0, 1 mM DTT, 1 mM EDTA, 70 mM KCl at 37°C for 10 min. Each value in C represents the average (±SD) of three independent measurements. ‘U-DNA incision (total)’ corresponds to the values obtained from measuring the strength of the bands on the gel in B (lanes 4–7); the ‘U-DNA incision (enzymatic)’ values are calculated by subtracting the amount of AP site incision caused by the 5-min heat treatment at 95°C (as presented in Figure 2D ) from the ‘U-DNA incision (total)’ values, where the number of AP sites formed by hSMUG1 equals the number of uracils excised as measured in parallel in B (lanes 8–10). Abbreviation: nt, nucleotides; UIP, U-DNA incision product; UPP, U-DNA processing product.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Excision Assay, Incubation

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: hSMUG1 incises at uracil in ssDNA. ( A, B ) Protein dependence of U-DNA incision (red) and uracil excision (blue). hSMUG1 was incubated with ssU-DNA (1 pmol; the labelled strand of substrate 1) at 37°C for 10 min. Each value in B represents the average of 2 independent measurements. Incision product was separated from un-incised DNA by PAGE at 100 V for 50 min using a 12% (w/v) gel with 3% (v/v) formamide.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Incubation, Polyacrylamide Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: hSMUG1 incises at uracil in DNA. ( A ) DNA substrate and assay. ( B, C ) Protein dependence of U-DNA incision (red) and uracil excision (blue). hSMUG1 was incubated with U-DNA (substrate 1, 1 pmol) at 37°C for 10 min. Each value in C represents the average (±SD) of 3–6 independent measurements. Incision product was separated from un-incised DNA by PAGE at 115 V for 1.5 h using a 20% (w/v) gel with 3% (v/v) formamide. ( D ) hSMUG1(25–270) was incubated with U-DNA (1 pmol of substrate 1; see A) at 37°C for 20 min. Incision product was separated from un-incised DNA by PAGE at 120 V for 2 h using a 20% (w/v) gel with 3% (v/v) formamide. ( E ) Protein dependence of U-DNA incision/processing (red) and uracil excision (blue). Each value represents the average (± SD) of 4–5 independent measurements as described in D. ( F ) U-DNA incision by hSMUG1 in different buffers. U-DNA (1 pmol of substrate 1) was incubated with 1 pmol of hSMUG1(25–270) or without enzyme as control in reaction buffer (HEPES), or in 45 mM sodium cacodylate with the same pH and additions as for reaction buffer (see Materials and Methods), at 37°C for 10 min (final volume, 20 μl). Incision product was separated from un-incised DNA by PAGE as described in E. Each value represents the average (±SD) of three independent measurements.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Incubation, Polyacrylamide Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: Indirect identification of UIP by electrophoretic mobility without exposure of DNA to high temperature. U-DNA (substrate 1, 1 pmol) was incubated with hSMUG1 (0.3 pmol) at 37°C for 30 min; either alone or together with EcFpg (4 pmol) as indicated. To define the different 3′-end products, substrate was incubated with hUNG (1 pmol) together with either EcFpg (4 pmol), hAPE1 (0.45 pmol) or EcNth (1 pmol), as indicated, under the same conditions. Incubations were also performed with either substrate 1 (dsDNA; lane 2) or the labelled strand of substrate 1 (ssDNA; lane 1) alone, showing that the upper substrate band is ssDNA and the lower band dsDNA. Incision product was separated from un-incised DNA by PAGE at 300 V for 5 h using a 20% (w/v) gel with 7 M urea.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Incubation, Polyacrylamide Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: Indirect identification of UIP and UPP by electrophoretic mobility using conventional denaturing conditions. ( A, B ) Time dependence of UIP (red) and UPP (green) formation by hSMUG1. hSMUG1 (0.3 pmol) was incubated with substrate 1[ 32 P] (0.12 pmol) in 20 mM Tris-HCl, pH 8.0, 1 mM DTT, 1 mM EDTA, 70 mM KCl at 37°C. To define the different 3′-end products, substrate was incubated with either EcNth (8.7 pmol), EcNfo (0.16 pmol), EcFpg (17 pmol) or hOGG1 (13 pmol) together with EcUng (0.78 pmol) for 10 min. Incised was separated from un-incised DNA by denaturing PAGE. Each value in B represents the average (±SD) of three independent measurements.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Incubation, Polyacrylamide Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: hSMUG1 kinetics. ( A ) Three-phase kinetic model. Phase 1 is shown in blue, phase 2 in violet and phase 3 in red. The uracil excision step is rapid compared to the slow DNA incision step. ( B ) U-DNA incision rate V in and ( C ) uracil excision rate V ex (see A) as a function of enzyme concentration [E] 0 at an initial U-DNA concentration [S] 0 of 50 nM, where the corresponding time-dependent data in the range [E] 0 = 0.05–0.25 nM (red line) is presented in ( D ) showing that at higher initial enzyme (E) concentration the model predicts that the formation of the incision product P1 has linear time-dependent kinetics, and in ( E ), showing that the excision kinetics for U (blue line) are fast and correlate with the removal of substrate DNA (S; black line), respectively. Incubation was performed for 20 min as described in Figure 4B . The V in in the blue area changes as a result of increased unspecific binding of enzyme to DNA. In the yellow area, the unspecific binding is saturated and the V in follows Michaelis–Menten (MM) kinetics. Each value represents the average (±SD) of 3–6 independent measurements.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Concentration Assay, Incubation, Binding Assay

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: Chemical identification of UIP and UPP and working model for reaction mechanism causing DNA incision. ( A ) Proposed E2 elimination reaction for the formation of UIP and chemical identification of UIP and UPP by MALDI-TOF-MS (see Supplementary Data, Figure S3 for MALDI-TOF-MS controls). hSMUG1 amino acid residue(s) suggested being involved in catalysis are coloured green; their hydrogen bonds with catalytic water and substrate are shown by red dotted lines. Proposed electronic and proton transfers involved in the formation of UIP are indicated by blue arrows. In the case of UPP, no reaction mechanism is proposed, and it is still unclear whether it is formed directly as a result of incision or by processing of UIP as depicted here. ( B ) Confirmation of the chemical nature of UIP. The observed post-enzymatic addition of water (left) or ammonia (middle and right) can be explained by the presence of a conjugated double bond, while the efficient exchange of an oxygen atom when the sample was transferred between 18 O- and 16 O-water can be explained by the presence of an aldehyde group. The MALDI-TOF-MS signals of the different chemical structures are shown in the upper and lower panels in A, and in the lower panel in B.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: Excision of uracil from DNA by hSMUG1 includes strand incision and processing

doi: 10.1093/nar/gky1184

Figure Lengend Snippet: Trapping experiments for Schiff base intermediate. Left panel, EcFpg (17 pmol) alone as a negative control, and together with EcUng (3 pmol) as a positive control, EcUng as well as hUNG (5 pmol) alone as negative controls, and hSMUG1 (0.3 pmol) alone, were incubated with substrate 2 (1 pmol) and 50 mM NaBH 4 in reaction buffer at 37°C for 1 h (final volume, 10 μl). Right panel, EcFpg (10 pmol) alone as a negative control, and together with EcUng (10 pmol) as a positive control, EcUng as well as hUNG (10 pmol) alone as negative controls, and hSMUG1 (10 pmol) alone, were incubated with substrate 2 (1 pmol) and 50 mM NaBH 4 in reaction buffer at 37°C for 1 h (final volume, 10 μl). In each case (A and B), trapped was separated from un-trapped substrate by denaturing PAGE [10% (w/v)] at 200 V for 1 h. The experiments were performed in triplicate showing the same result.

Article Snippet: Enzymes hSMUG1 (full length) was obtained from NEB (New England BioLabs) and investigated for contaminants by MS analysis (see ) as well as purified by us [see , Production of purified hSMUG1(25–270) and ].

Techniques: Negative Control, Positive Control, Incubation, Polyacrylamide Gel Electrophoresis