A poxvirus-like type IB topoisomerase family in bacteria

Abstract

We report that diverse species of bacteria encode a type IB DNA topoisomerase that resembles vaccinia virus topoisomerase. Deinococcus radiodurans topoisomerase IB (DraTopIB), an exemplary member of this family, relaxes supercoiled DNA in the absence of a divalent cation or ATP. DraTopIB has a compact size (346 aa) and is a monomer in solution. Mutational analysis shows that the active site of DraTopIB is composed of the same constellation of catalytic side chains as the vaccinia enzyme. Sequence comparisons and limited proteolysis suggest that their folds are conserved. These findings imply an intimate evolutionary relationship between the poxvirus and bacterial type IB enzymes, and they engender a scheme for the evolution of topoisomerase IB and tyrosine recombinases from a common ancestral strand transferase in the bacterial domain. Remarkably, bacteria that possess topoisomerase IB appear to lack DNA topoisomerase III. The type IB family of DNA topoisomerases includes eukaryotic nuclear topoisomerase I and the topoisomerases of poxviruses (1, 2). The type IB enzymes relax DNA supercoils via a multistep reaction pathway entailing noncovalent binding of the enzyme to duplex DNA, cleavage of one DNA strand with formation of a covalent DNA-(3Ј-phosphotyrosyl)-protein intermediate, strand passage, and strand religation. A constellation of conserved amino acid side chains (Arg-130, Lys-167, Lys-220, Arg-223, and His-265 in vaccinia virus topoisomerase) catalyzes the attack of the active site tyrosine nucleophile (Tyr-274) on the scissile phosphodiester. The RKKRH ''catalytic pentad'' is conserved in all members of the topo IB family. The Arg-130, Lys-167, Arg-223, and His-265 components of the pentad each lend a 10 2 -10 5 enhancement of the transesterification rate (3-8). The Int family of site-specific DNA recombinases (now called tyrosine recombinases) use a similar mechanism to catalyze formation and resolution of Holliday junctions. The catalytic pentad of the tyrosine recombinases (RKHRH) characteristically contains a histidine at the third position occupied by lysine in topo IB (9).topo IB and tyrosine recombinases consist of two domains that form a C-shaped protein clamp around duplex DNA. The carboxyl-terminal catalytic domains of topo IB and tyrosine recombinases adopt a common fold composed of eight ␣-helices and a three-strand antiparallel ␤-sheet (6, 10-15). The constituents of the catalytic pentad occupy similar positions in the tertiary structures of topo IB and tyrosine recombinases. Thus, it is proposed that topo IB and tyrosine recombinases evolved from a common ancestral DNA strand transferase (11), in which case we can consider them distinct branches of an enzyme superfamily defined by a DNA-3Ј-phosphotyrosyl intermediate.Remarkably, there is no structural similarity at all between the amino-terminal domains of type IB topoisomerases and those of the tyrosine recombinases. For example, the amino-terminal domain of vaccinia topoisomerase consists almost entirely of ␤-strands, whereas the amino t...

New Peptide Inhibitors of Type IB Topoisomerases: Similarities and Differences Vis-a-vis Inhibitors of Tyrosine Recombinases

“…The amount of remaining supercoiled substrate was compared among the treated reactions ( Fig. 1(b), lanes [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] and to untreated control reactions (Fig 1(b), lanes 1 and 2). In the example shown, the mixtures containing amino acids C, F, L, M, W and Y at the second position inhibit vTopo activity most.…”

Drosophila topo IIIα is required for the maintenance of mitochondrial genome and male germ-line stem cells

“…There are two isozymes of topo III, α and β, in metazoans: topo IIIα is essential for viability, and topo IIIβ is not (3,4). Eucaryotic topo I is classified as type IB enzymes, and it forms an evolutionarily distinct class when compared with type IA enzymes, presumably a descendent of site-specific recombinases in bacteria (5). These enzymes work by generating a strand break where protein is linked to the 3 0 -phosphoryl end, allowing the free 5 0 end to undergo a restrained rotation around the nonscissile strand (6).…”

Characterization of Mimivirus DNA Topoisomerase IB Suggests Horizontal Gene Transfer between Eukaryal Viruses and Bacteria

“…Mimivirus, poxvirus, and bacterial TopIB proteins are of similar size (314 to 417 aa). Human TopIB (a 765-aa polypeptide) and other nuclear TopIB enzymes are much larger and contain multiple structural modules added on to a protein scaffold that is otherwise quite similar to that of the vaccinia TopIB (5,6,15). It has been proposed that nuclear TopIB enzymes evolved from a bacterial or poxvirus-like precursor (15).…”

“…MimiTopIB joins a growing TopIB enzyme family that includes eukaryotic nuclear and mitochondrial TopIB (41), poxvirus topoisomerases, and poxvirus-like topoisomerases encoded by the genomes of many bacteria (15). Mimivirus, poxvirus, and bacterial TopIB proteins are of similar size (314 to 417 aa).…”

“…Mimivirus is a parasite of the protozoan Acanthamoeba polyphaga, and virtually nothing has been published about the viral replication cycle. The putative mimivirus type IB topoisomerase (MimiTopIB) displays primary structure similarity to poxvirus topoisomerases and, to an even greater extent, to the recently discovered family of bacterial poxvirus-like TopIB enzymes (15) (Fig. 1).…”