Characterization of DNA Binding by the Isolated N-Terminal Domain of Vaccinia Virus DNA Topoisomerase IB

Abstract

Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal-type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5'-(T/C)CCTT↓, leading to the formation of a covalent DNA-(3'-phosphotyrosyl²⁷⁴)-enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81-314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with a cleavage site affinity lower than that of the full-length enzyme. The N-terminal domain (amino acids 1-80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in the DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ∼2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution nuclear magnetic resonance methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB is underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength.

Figure 1.

(A) Schematic representation of contacts between TopN and a segment of the 24-mer DNA duplex containing the 5’-CCCTT-3’ consensus sequence (spDNA). The grey circles represent the nucleosides of the scissile strand and orange circles represent those of the non-scissile strand; red circles represent backbone phosphate groups. Hydrophobic contacts and hydrogen bonds are represented by solid and dashed arrows, respectively. The scissile phosphate, represented by the blue circle, lies between the +1T and −1A nucleosides. The details of the contacts are summarized in Table S1. Bases that have been altered to generate the nsDNA duplex from the spDNA duplex are shown with a red (instead of a black) outline. Selected sidechain contacts made by TopN with the non-scissile strand (B) and the scissile strand (C) are shown as stereo images from PDB ID: 3IGC. Key residues and nucleobases are numbered.

Figure 2.

Measurement of the binding of TopN to nsDNA (A) and spDNA (B) duplexes via isothermal calorimetry (ITC). The data shown are representative of duplicate measurements. DNA duplexes used are shown at the top of the corresponding ITC traces. The bases of the specific CCCTT (underlined) sequence on the spDNA (red lettering) that have been altered to generate the nsDNA (blue lettering) are indicated. The arrow indicates the site of cleavage by vTopIB. The binding affinity (K_D) and the corresponding ΔH and ΔS values obtained from the fits of the data represented are also indicated.

Figure 3.

Assigned ¹⁵N, ¹H HSQC (600 MHz) of TopN. Backbone assignments are labeled in black and sidechain assignments are labeled in red.

Figure 4.

(A) Chemical shift perturbations (CSPs) in TopN in the presence of two molar equivalents of either spDNA (black) or nsDNA (red). Titrations were carried out under low salt conditions in NMR buffer (see text). Regions encompassing secondary structure elements are shaded and labeled. The C-terminal segment (shaded cyan, labeled α3) is helical only in the context of the full-length structure in complex with DNA. Amino acids whose sidechains make contact with DNA in the structure of the transition-state mimic of full-length vTopIB are indicated by (*). (B) For spatial representation, the CSPs in the presence of spDNA (top) and nsDNA (bottom) have been mapped onto the surface of the N-terminal domain in the crystal structure of vTopIB bound to the consensus sequence. Key residues that display significant perturbations are labeled. The C domain has been omitted for clarity.

Figure 5.

(A) Differences in chemical shifts for TopN in the presence of two molar equivalents of spDNA or nsDNA. Resonances in the presence of spDNA were used as reference to calculate the perturbations using Equation 1. Residues with the largest differences are shown in red. (B) Residues with significant differences in resonance positions in the presence of spDNA or nsDNA are mapped onto the surface of the N domain in the crystal structure of of vTopIB bound to the consensus sequence, and colored red. H39 and L40 that exhibit the most significant differences are labeled in larger font. The C domain has been omitted for clarity.

Figure 6.

Δ_norm,salt values calculated using Equation 2 for spDNA (open black bars) and nsDNA (closed red bars) plotted against residue number.

Figure 7.

Effect of double-alanine mutations on DNA cleavage. (A) Aliquots (4 μg) of the phosphocellulose preparations of wild-type vTopIB and the indicated double-mutants 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) Single-turnover DNA cleavage reactions were performed as described under MATERIALS AND METHODS. The 5′-³²P-labeled 18-mer/30-mer DNA substrate is depicted at the top with the 5′-³²P-label denoted by • and the topoisomerase cleavage site indicated by an arrow. The apparent cleavage rate constants (k_cl±SE) were calculated by nonlinear regression of the kinetic data to a one-phase association model. The asterisk denotes a mutation that elicits a >10-fold decrease in cleavage rate.

Figure 8.

Salt-sensitivity of DNA cleavage. The extents of covalent complex formation normalized to that of a control reaction without added salt are plotted as a function of NaCl concentration for wild-type vTopIB and double-alanine mutants. Each datum is the average of two independent experiments.