The interaction between transport-segment DNA and topoisomerase IA—crystal structure of MtbTOP1 in complex with both G- and T-segments

Abstract

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.

What’s on the Other Side of the Gate: A Structural Perspective on DNA Gate Opening of Type IA and IIA DNA Topoisomerases

“…A crystal structure of M. tuberculosis TopI bound to the G-segment and the DNA trapped within the toroid hole was recently published and proposed to represent the state immediately following the G-strand re-ligation [ 56 ] ( Figure 3 b, state 5a). However, since only one DNA oligonucleotide was used to form the complex, the trapped T-segment is a non-canonical parallel duplex that formed as a result of crystal packing.…”

“…In type IA enzymes, the amino acid sequence of domain DII, which comprises the largest part of the toroid hole, is less conserved compared to the rest of the catalytic core [ 82 ]. Even though they generally do have positively charged residues facing the inner side of the central cavity, their number and distribution vary depending on species and subtypes [ 56 , 82 ]. This points to the absence of specific protein-DNA interactions within the hole.…”

“…Both would result, at least to some degree, in a reduction of the effective size of the central cavity. Currently, it is unknown what the roles of these loops are, but being flexible elements, they might regulate the strand passage and/or gate opening [ 56 , 85 ]. The available structural as well as biophysical data suggested that type IA enzymes have sufficient plasticity to widen the central hole to accept double-stranded DNA [ 56 , 83 ].…”

“…Over a dozen full-length or partial crystal structures of IA topoisomerases from various organisms with or without bound DNA have been solved to date, but these structures have not provided information on the conformational changes that open the DNA-gate and promote T-segment passage [ 51 , 56 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 68 , 70 , 71 , 80 , 81 , 85 , 86 ]. One exception might be the structure of the isolated fragment of the DII/DIII domains from E. coli TopI [ 87 ] ( Figure 3 b, state 3b).…”

“…The enzyme is believed to capture the T-segment within the central hole, which is capable of accommodating dsDNA [ 54 , 55 ]. The binding of a double-stranded T-segment is likely accompanied by a conformational change that results in widening of the toroid hole [ 56 ] (4a). Alternatively, partial DNA-gate opening might already provide an opening that is big enough for passage of single-stranded DNA, leading to supercoil relaxation [ 53 ] (4b).…”