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1.
Figure 2

Figure 2. Chemical mutagenesis of vaccinia virus TopIB Lys167. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

The mutagenesis strategy is outlined. (A) Aliquots (5 μg) of the indicated phosphocellulose preparations of vaccinia TopIB were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) Chemical reaction of 2-bromoethylamine with cysteine to form γ-thialysine. (C) Chemical structures of other bromoalkyl compounds tested.

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.
2.
Figure 1

Figure 1. Structural basis of TopIB transesterification chemistry. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

The active sites of the pentavalent vanadate complex of poxvirus TopIB (a transition state mimetic; PDB 3IGC; shown at left) and the covalent tyrosyl-DNA intermediate of poxvirus TopIB (PDB 2H7F; shown at right) were superimposed and then offset laterally. The catalytic amino acid side chains are indicated. Atomic contacts are denoted by dashed lines.

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.
3.
Figure 4

Figure 4. Tests of chemical rescue of K167C by haloalkyl compounds. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

Single-turnover cleavage by mock-treated and haloalkyl-treated TopIB proteins was performed as described in the legend to Fig. 3. The extents of covalent complex formation were quantified by scanning the dried SDS gels with a Fujifilm BAS-2500 imager. Each datum in the bar graph is the average of three independent experiments ± SEM. The alkylating agents were: 2-bromoethylamine (2-BEA), 3-bromoprolylamine (3-BPA), 2-bromoethanol (2-BE), and 2-bromoacetate (2-BA).

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.
4.
Figure 8

Figure 8. Aminoalkylation of Cys167 restores supercoil relaxation activity. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

Relaxation reaction mixtures containing (per 20 μl) 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 300 ng of supercoiled pUC19 DNA, and 2.25 ng of WT, K167C, 2-BEA-treated K167C or 3-BPA-treated K167C TopIB as specified were incubated at 37°C. The reactions were initiated by the addition of enzyme. Aliquots (20 μl) were withdrawn at the times specified and quenched immediately with SDS. The time 0 samples were taken prior to addition of enzyme. The reaction products were analyzed by 1% agarose horizontal gel electrophoresis. The gels were stained with ethidium bromide and DNA was visualized under UV illumination.

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.
5.
Figure 6

Figure 6. Kinetics of single-turnover cleavage by 2-BEA- and 3-BPA-modified TopIB. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

(A) Kinetic analysis of single turnover cleavage of the 18-mer/30-mer substrate was performed as described under Experimental Procedures. The percentage of input DNA cleaved was plotted as a function of reaction time for the indicated TopIB preparations. Each datum in the graph is the average of three independent time course experiments ± SEM. The cleavage rate constants were derived by nonlinear regression fitting of the data to an exponential one-phase association in Prism. (B) Cleavage reactions mixtures containing 0.3 pmol 5′ 32P-labeled 18-mer/30-mer DNA substrate and increasing amounts of TopIB as specified were incubated for 15 min at 37°C. The percentage of input DNA cleaved was plotted as a function of input TopIB. Each datum in the graph is the average of three independent protein titration experiments ± SEM. (C) The kinetics of single-turnover cleavage of 0.3 pmol 5′ 32P-labeled 18-mer/30-mer DNA substrate by 150 ng (4 pmol) of either pure wild-type TopIB (WT), pure K167C, 2-BEA-treated K167C, or a 25%/75% mixture of WT and K167C.

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.
6.
Figure 3

Figure 3. Aminoalkylation of Cys167 restores DNA cleavage activity. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

The single-turnover cleavage reaction is depicted at the bottom of the figure. The 18-mer/30-mer suicide cleavage substrate is shown with the 5′ 32P-label denoted by ●. The poxvirus TopIB forms a C-shaped protein clamp around the CCCTTp↓A recognition site. Transesterification results in the formation of a covalent 32P-labeled pCGTGTCGCCCTTp-(TopIB) adduct and dissociation of the unlabeled HOATTCCC leaving strand. Cleavage reaction mixtures (20 μl) containing 50 mM Tris-HCl (pH 7.5), 0.3 pmol 5′ 32P-labeled DNA substrate and 150 ng (4 pmol) of TopIB as specified (by numbers at left and above the gel lanes) were incubated at 37°C for 10 min. The reactions were quenched with 1% SDS. The products were analyzed by SDS-PAGE. An autoradiogram of the dried gel is shown. Free DNA migrated near the dye front. Covalent complex formation was revealed by transfer of radiolabeled DNA to the TopIB protein. The arrows overlying lanes 3 highlight the successful chemical rescue of K167C DNA cleavage activity by 2-bromoethylamine and 3-bromopropylamine.

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.
7.
Figure 5

Figure 5. Kinetics of equilibrium DNA cleavage by K167A and K167C. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

The 34-mer/30-mer equilibrium cleavage substrate is depicted at the top with the 5′ 32P-label denoted by ●, the CCCTT sequence shaded gray, and the cleavage site indicated by the arrow. Reaction mixtures containing (per 20 μl) 50 mM Tris-HCl (pH 7.5), 0.3 pmol of 32P-labeled DNA, and 150 ng (4 pmol) of purified K167A or K167C TopIB were incubated at 37°C. The reactions were initiated by adding TopIB to pre-warmed reaction mixtures. Aliquots (20 μl) were withdrawn at the times specified and the reactions were quenched immediately with SDS. The quenched reactions were treated with 10 μg of proteinase K for 60 min at 37°C, then mixed with an equal volume of 95% formamide/20 mM EDTA, and heat-denatured prior to electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE (90 mM Tris-borate, 2.5 mM EDTA). The radiolabeled DNAs and the DNA-peptide adducts derived by proteolysis of the covalent TopIB-DNA complex were quantified by scanning the gel. Covalent adduct formation (expressed as the percent of the total labeled DNA) is plotted as a function of reaction time. Each datum is the average of three separate experiments ±SEM. Non-linear regression curve fits to a pseudo-first order exponential function (executed in Prism) are shown.

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.
8.
Figure 7

Figure 7. Effects lysine analogs on DNA religation. From: Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

Cleavage reaction mixtures containing (per 20 μl) 50 mM Tris-HCl (pH 7.5), 0.3 pmol 5′ 32P-labeled 18-mer/30-mer DNA substrate, and 150 ng (4 pmol) of TopIB as specified were incubated at 37°C for 5 min to form the suicide intermediate depicted at the top of the figure. Religation was initiated by the simultaneous addition of NaCl to 0.5 M and a 5′-OH 18-mer acceptor strand d(ATTCCGATAGTGACTACA) to a concentration of 15 pmol/22 μl (a 50-fold molar excess over the input DNA substrate). Aliquots (20 μl) were withdrawn at the times specified and quenched immediately with 1% SDS. A time 0 sample was withdrawn before the addition of the acceptor strand. The samples were digested for 1 h at 37°C with 10 μg of proteinase K, then mixed with an equal volume of 95% formamide/20 mM EDTA, heat-denatured, and analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. The radiolabeled DNAs and DNA-peptide adducts (derived by proteolysis of the covalent TopIB-DNA complex) were quantified by scanning the gel. Religation of the covalently bound 12-mer strand to the 18-mer acceptor DNA yielded a 5′ 32P-labeled 30-mer strand transfer product. The extents of religation (expressed as the percent of the covalent intermediate converted into 30-mer) are plotted as a function of reaction time for each TopIB protein. Each datum for the alkylated K167C proteins is the average of three separate experiments ±SEM. Each datum for wild-type TopIB is the average of five experiments ±SEM.

Lyudmila Yakovleva, et al. Biochemistry. 2013 February 5;52(5):984-991.

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