Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion

Abstract

Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB-DNA complexes has suggested that the enzyme promotes long-range DNA-DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg2+, Ca2+ or Mn2+, suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge.

Figure 1

Control experiments. DNA molecules are not observed to cluster in the absence of vTopIB when adsorbed on a mica surface using MgCl₂ (a) or polylysine-coated mica (b). The appearance of the DNA molecules differs as a result of the adsorption processes involved. (c) Histogram of the height of vTopIB molecules measured in buffer in the absence of DNA. The average height of vTopIB in solution is 3.6 ± 0.9 nm, compatible with a monomeric state of the protein. An AFM image of vTopIB in buffer is shown as an inset in (c). Bar size is 200 nm. (d) Summary of cooperativity theory (19,20). Three different binding events can take place: isolated binding with an association constant K (d, i), singly contiguous binding with an association constant Kω (d, ii) and doubly contiguous binding with association constant Kω² (d, iii). In our model an isolated binding event is attributed to the formation of an intramolecular node.

Figure 2

vTopIB–DNA complexes at low vTopIB:DNA values. The concentration of DNA is constant in all experiments and equal to 0.22 nM. (a) Gallery of AFM images of vTopIB bound to linear DNA. Three different types of complexes were found. (a, i) Individual vTopIB proteins bound to DNA ([vTopIB] = 4 nM), nodes (a, ii) and filament-like structures (a, iii) ([vTopIB] = 6.2 nM). (b) Gallery of AFM images of vTopIB bound to nicked-circular DNA. Similar types of complexes were found. (b, i) Individual vTopIB–DNA complexes ([vTopIB] = 4 nM), nodes (b, ii) and filament-like structures (b, iii) ([vTopIB] = 13.4 nM). Bar size is 100 nm.

Figure 3

Intermolecular synapsis of DNA at high vTopIB:DNA values. The concentration of DNA is constant in all experiments and equal to 0.22 nM. (a) AFM images of linear and circular DNA molecules incubated with [vTopIB] = 12.4 nM and [vTopIB] = 45 nM, respectively, showed clustering in the presence of Mg²⁺ cations (top). This effect was not cation-specific, as incubation of linear DNA and 12.4 nM [vTopIB] together with Ca²⁺ or Mn²⁺ likewise resulted in the formation of aggregates. Bar size is 100 nm. (b) Intermolecular synapsis of DNA was quantified by counting the number of DNA molecules involved in an cluster at different vTopIB:DNA ratios in the presence of MgCl₂. The fraction of DNA molecules in a cluster increased linearly with increasing vTopIB concentration and saturated at very high vTopIB concentrations. This saturation was due to an underestimation of the number of DNA molecules involved (see text for details).

Figure 4

Intermolecular synapsis of DNA is affected by the presence of MgCl₂. (a) Linear DNA molecules were deposited on polylysine-coated mica and incubated with vTopIB in the absence of MgCl₂ again resulting in the observation of vTopIB-bound DNA (white arrows). Under these conditions, however, the DNA molecules did not cluster. (b) Linear DNA molecules deposited on polylysine-coated mica showed intermolecular synapses when incubated with vTopIB and MgCl₂. Bar size is 200 nm.

Figure 5

Model describing the formation of filaments of vTopIB on DNA. The constituents of the filaments may be vTopIB dimers (a) or monomers (b). The formation of filaments requires several steps. (i) The formation of an intramolecular node with an association constant K^*. (ii) The node is extended by binding of new vTopIB protomers with an association constant of K^*ω. (iii) A filament of length c is generated.

Figure 6

Quantitative analysis of the binding of vTopIB to DNA. (a) Filament length distribution for three different data sets. For each set ω was estimated using the ML method resulting in ω = 5.6 ± 1.4 × 10³ for DNA saturation θ = 0.07 (closed squares); ω = 9.1 ± 1.7 × 10³ for DNA saturation θ = 0.18 (closed circles); and ω = 8.8 ± 1.8 × 10³ for DNA saturation θ = 0.23 (closed triangles). The solid lines in (a) are plots of Equation 1 using the obtained ω. (b) Fractional DNA saturation as a function of total vTopIB concentration. The fit of Equation 4 to these data using n = 18, N = 2743 and ω = 7.7 ± 1.1 × 10³ yielded a value for K^* = 4.0 ± 0.4 × 10⁴ M⁻¹.