Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5′-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

Each catalytic cycle of type IA topoisomerases has been proposed to comprise multistep reactions. The capture of the transport-segment DNA (T-segment) into the central cavity of the N-terminal toroidal structure is an important action, which is preceded by transient gate-segment (G-segment) cleavage and succeeded by G-segment religation for the relaxation of negatively supercoiled DNA and decatenation of DNA. The T-segment passage in and out of the central cavity requires significant domain–domain rearrangements, including the movement of D3 relative to D1 and D4 for the opening and closing of the gate towards the central cavity. Here we report a direct observation of the interaction of a duplex DNA in the central cavity of a type IA topoisomerase and its associated domain–domain conformational changes in a crystal structure of a Mycobacterium tuberculosis topoisomerase I complex that also has a bound G-segment. The duplex DNA within the central cavity illustrates the non-sequence-specific interplay between the T-segment DNA and the enzyme. The rich structural information revealed from the novel topoisomerase–DNA complex, in combination with targeted mutagenesis studies, provides new insights into the mechanism of the topoisomerase IA catalytic cycle.

Type IA topoisomerases relax negatively supercoiled DNA by catalyzing the breaking and rejoining of DNA single‐strand coupled with DNA strand passage through the break. The proposed mechanism suggested that the torus‐shaped structure formed by domains D1‐D4 of the enzymes can adopt two possible conformations: “closed” and “open” with D3 in contact with D1 and proximity to D4 or D3 moving away from D1 and D4 to open up the toroid hole. However, the molecular basis of the opening‐closing of the toroid hole remains elusive. We hypothesized that the perturbation of any interaction between D3 and D4 in the interface would make the enzyme‐bridged gate opening easier or closing harder. We selected some conserved residues based on the structure and sequence alignment of type IA topoisomerases in the interface between D3 and D4 that might play a role in interdomain communication. These conserved residues in E. coli topoisomerase I (EcTopI) include S287 and Q290 in D3 and E487 in D4. Site‐directed mutagenesis was used to create alanine substitutions at these polar residues. Growth complementation assay using AS17 strain with temperature‐sensitive topoisomerase I showed a ten‐fold reduction in growth complementation for EcTopI‐E487A compared to wild‐type EcTopI. Six‐fold reduction in relaxation activity was also observed for EcTopI‐E487A. Our findings suggest that strictly conserved residues that establish the interaction of D3 and D4 of E. coli topoisomerase I can play an important role in enzyme conformational changes. The focus of this study could provide more in‐depth insights into the mechanism of type IA topoisomerases.

E coli topoisomerase I (EcTopI) is a type IA topoisomerase that relaxes negatively supercoiled DNA to prevent the inhibition of vital cellular processes such as transcription. Type IA topoisomerase polypeptide folds into a torus structure to catalyze the breaking and rejoining of a DNA single‐strand coupled with DNA strand passage, thus maintain the DNA topology. Previously elucidated full‐length crystal structure of EcTopI consisting nine domains (D1–D9), bound with an ssDNA to the C‐terminal domain revealed that D6 has an unusual 21‐amino acids insert in the middle of the first β‐strand. A part of the insert (12 amino acids) forms a unique alpha helix (α1), which is present in the vicinity of the N‐terminal domain at the hinge region of D2 and D4. We hypothesized that the C‐terminal element D6 domain interacts with the N‐terminal elements D2 and D4 domains hinge region through this α1‐helix and aids in the separation of D3 from D4 and D1, thus creating an opening to the toroid hole for DNA strand passage in the catalytic cycle. Site‐directed mutagenesis has been employed to create two mutant EcTopI proteins with deletion of D6‐ α1 helix (residues 647–658, Mut‐1) and substitutions of A651G and A655G to perturb α1‐helix structure (Mut‐2). Biochemical analysis of these mutant proteins showed significantly reduced relaxation activity with negatively supercoiled DNA as substrate, and the kinetics of DNA negative supercoil removal by mutants EcTopI showed clear decrease in the rate of relaxation at different time points versus wild‐type EcTopI. To test the effect of these mutations on protein’s thermal stability, thermal shift assay has been carried out and the result suggested that the mutations affected the inter‐domain interactions without significant destabilization of the polypeptide structure as only the first melting peak is shifted to lower temperature but no shift in second peak has been observed compared to wild‐type. Our findings suggest that D6 α1‐helix supports a functional role in the hinge regions between D2 and D4 where its movement (e.g. a push or a pull) as an arm against the hinge will cause the rotation of D2 in respect to D4 leading to the enzyme undergoing a large conformational change required for catalysis. The results of this study have provided a more in‐depth mechanistic insight into the mechanism of type IA topoisomerases that is required for maintaining DNA topology. Support or Funding Information NIH grant R 01 GM 054226 E coli topoI‐Crystal Structure Thermal shift assay