Characterization of mimivirus DNA topoisomerase IB suggests horizontal gene transfer between eukaryal viruses and bacteria

Abstract

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes dozens of proteins with imputed functions in nucleic acid transactions. Here we produced, purified, and characterized mimivirus DNA topoisomerase IB (TopIB), which we find to be a structural and functional homolog of poxvirus TopIB and the poxvirus-like topoisomerases discovered recently in bacteria. Arginine, histidine, and tyrosine side chains responsible for TopIB transesterification are conserved and essential in mimivirus TopIB. Moreover, mimivirus TopIB is capable of incising duplex DNA at the 5'-CCCTT cleavage site recognized by all poxvirus topoisomerases. Based on the available data, mimivirus TopIB appears functionally more akin to poxvirus TopIB than bacterial TopIB, despite its greater primary structure similarity to the bacterial TopIB group. We speculate that the ancestral bacterial/viral TopIB was disseminated by horizontal gene transfer within amoebae, which are permissive hosts for either intracellular growth or persistence of many present-day bacterial species that have a type IB topoisomerase.

FIG. 1.

Mimivirus TopIB. The amino acid sequence of the mimivirus (Mimi) TopIB-like protein is aligned with the poxvirus-like bacterial TopIB polypeptides encoded by genes of Pseudomonas putida (Ppu), Xanthomonas campestris (Xca), Bordetella parapertussis (Bpa), Deinococcus radiodurans (Dra), and Mycobacterium avium (Mav). Gaps in the alignment are indicated by dashes. Positions of side chain identity and/or similarity in all six proteins are indicated by dots. The conserved equivalents of the active site of vaccinia virus TopIB are highlighted in shaded boxes.

FIG. 2.

Purification and DNA cleavage activity of MimiTopIB. (A) Aliquots (5 μg) of the tag-free wild-type (WT) MimiTopIB and the R135A, R241A, H285A, and Y294A mutants were analyzed by SDS-PAGE in parallel with recombinant vaccinia virus TopIB (Vac). The Coomassie blue-stained gel is shown. The positions and sizes (in kilodaltons) of marker polypeptides are indicated on the left. (B) Reaction mixtures (20 μl) containing 50 mM Tris-HCl, pH 7.5, 0.3 pmol 5′-labeled 18-mer/30-mer suicide DNA substrate (depicted at bottom, with the 5′ label indicated by the dot and the presumptive cleavage site denoted by the arrow) and increasing amounts of vaccinia or mimivirus TopIB (75, 150, 300, 450, or 600 ng, proceeding from left to right) were incubated at 37°C for 10 min. TopIB was omitted from control reaction mixtures in lanes labeled with dashes. The reactions products were resolved by SDS-PAGE. An autoradiograph of the gel is shown. The positions of the Topo-DNA adducts and the free DNA are indicated on the right.

FIG. 3.

Supercoil relaxation activity of MimiTopIB. (A) The left panel shows reaction mixtures (20 μl) containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.3 μg pUC19 DNA, and 0, 10, 20, 30, 40, or 50 ng of wild-type MimiTopIB incubated for 30 min at 37°C. The right panel shows a reaction mixture (160 μl) containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2.4 μg pUC19 DNA, and 200 ng of wild-type protein incubated at 37°C. Aliquots (20 μl) were withdrawn at the times specified. The DNA products were resolved by agarose gel electrophoresis and stained with ethidium bromide. The positions of supercoiled (SC) and relaxed (Rel) circular DNAs are indicated on the right. (B) Reaction mixtures (160 μl) containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2.4 μg pUC19 DNA, and 400 ng of the indicated MimiTopIB-Ala proteins were incubated at 37°C. Aliquots (20 μl) were withdrawn at the times specified.

FIG. 4.

Glycerol gradient sedimentation. MimiTopIB was sedimented through a 15 to 30% glycerol gradient as described in Materials and Methods. (A) Aliquots (18 μl) of the odd-numbered gradient fractions were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The polypeptides corresponding to MimiTopIB and marker proteins catalase, BSA, and cytochrome c (cyt c) are indicated by arrowheads flanking the gel. (B) DNA relaxation activity. Reaction mixtures (20 μl) containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.3 μg pUC19 DNA, and 2.5 μl of the odd-numbered gradient fractions were incubated at 37°C for 10 min. The DNA products were resolved by agarose gel electrophoresis and stained with ethidium bromide. (C) DNA cleavage activity. Reaction mixtures (20 μl) containing 50 mM Tris-HCl, pH 7.5, 0.3 pmol ³²P-labeled 18-mer/30-mer suicide substrate, and 2.5 μl of the odd-numbered gradient fractions were incubated at 37°C for 15 min. Activity was quantified as the percentage of the input labeled material that was converted to a covalent TopIB-DNA complex.

FIG. 5.

Kinetic analysis of single-turnover cleavage and religation. (A) Single-turnover cleavage. The 5′ ³²P-labeled suicide substrate and the covalent topoisomerase-DNA adduct are depicted at the top of the figure. Reaction mixtures (160 μl) containing 50 mM Tris-HCl, pH 7.5, 2.4 pmol 18-mer/30-mer DNA, and 3.6 μg of wild-type or H285A MimiTopIB were incubated at 37°C. Aliquots were withdrawn at 0, 5, 10, 20, 30, 60, 120, and 300 s for the wild-type (WT) MimiTopIB and at 0, 5, 10, 20, 30, and 60 min for the H285A mutant and quenched immediately by adding SDS. The percentage of the total radiolabel in the covalent TopIB-DNA complex is plotted as a function of time. (B) Single-turnover religation. The suicide covalent intermediate, the 18-mer acceptor strand, and the religation reaction product are depicted at the top of the figure. The suicide cleavage complex was formed during a 30-min preincubation at 37°C as described in Material and Methods. Religation was initiated by the simultaneous addition of NaCl to 0.25 M and a 100-fold molar excess of the 18-mer acceptor strand. Aliquots were withdrawn at the times specified. The extent of religation is plotted as a function of time postaddition of the acceptor strand.

FIG. 6.

Equilibrium cleavage. A 5′ ³²P-labeled 34-mer scissile strand, containing either a standard Tp↓A phosphodiester or a 5′-bridging phosphorothiolate Tp↓(S)A at the presumptive cleavage site, was hybridized to a sevenfold molar excess of the complementary 60-mer strand to form the equilibrium cleavage substrate shown at the top of the figure. The cleavage site is indicated by the arrow. Cleavage reactions were performed as described in Materials and Methods. The extent of covalent adduct formation is plotted as a function of input MimiTopIB protein.