Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

Patel, Asmita V.; Shuman, Stewart; Mondragón, Alfonso

doi:10.1074/jbc.m512332200

Cited by 29 publications

(36 citation statements)

References 63 publications

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“…Another type IB topoisomerase with strong similarities to the viral enzymes is the bacterial type IB enzyme from Deinococcus radiodurans (17,18). A step-size distribution for D. radiodurans topo IB was monitored at a force of 1 pN in the absence of glycerol, giving a mean step size of 54.4 Ϯ 33.8 turns.…”

Section: Resultsmentioning

confidence: 99%

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Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

Taneja

Schnurr

Slesarev

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or IB enzymes. We used real-time single-molecule micromechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type IB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type IB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type IB molecules but by using a completely different structural and mechanistic framework.magnetic tweezers ͉ single molecule ͉ archaeon ͉ DNA topology ͉ type IC T opoisomerase V (topo V) of the archaeon Methanopyrus kandleri is a 110-kDa enzyme belonging to the type I family of topoisomerases (1). Type I topoisomerases previously have been subdivided into two subtypes, IA and IB (2), based on whether they form a transient 5Ј or 3Ј covalent bond with the broken DNA strand (reviewed in ref.3). Topo V possesses biochemical properties associated with type IB topoisomerases, namely, cleavage of a single DNA strand, formation of a covalent intermediate with the 3Ј end of the broken strand, and the ability to relax both positive and negative supercoils in a magnesium-and ATP-independent manner (1). Despite these commonalities, topo V shows no sequence similarity to other type IB enzymes (4). Furthermore, the structure of topo V bears no resemblance to any known topoisomerase but instead reveals a unique fold (5). Although the active site of topo V includes a constellation of amino acids similar to that in type IB enzymes, their spatial arrangement is different. All these data suggest that, despite overall similarities, topo V and type IB enzymes use different catalytic mechanisms for DNA cleavage and religation. Furthermore, the mechanism of DNA relaxation used by topo V is unknown.Two mechanisms of DNA relaxation have been discussed for type I topoisomerases: an enzyme-bridged strand passage mechanism and a swiveling or ''controlled rotation'' mechanism. Type IA topoisomerases have a characteristic toroidal shape (6) and use an enzyme-bridged strand passage mechanism where both ends of the broken DNA strand are bound to the enzyme, preventing DNA swiveling. The intact DNA strand is passed through the transient break in the other strand before the topoisomerase religates the broken strand. This mechanism changes the DNA linking number strictly in increments of one (⌬Lk ϭ Ϯ1) (7-9). Type IB enzymes use a radically different mechanism involving swiveling, or rotation of one DNA strand around the other, to release supercoils. The protein embraces the DNA and cleaves one strand, allowing the free cleav...

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Section: Resultsmentioning

confidence: 99%

“…D. radiodurans topo IB and vaccinia topo IB enzymes were used as controls for comparisons with topo-78. D. radiodurans topo IB was purified as described (17,18), and vaccinia virus topo IB was obtained from Epicentre Biotechnologies, Madison, WI.…”

Section: Methodsmentioning

confidence: 99%

Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

Taneja

Schnurr

Slesarev

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Four conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265) are responsible for catalyzing the attack of the active site tyrosine (Tyr-274) on the scissile phosphodiester to form the covalent intermediate (2)(3)(4)(5)(6). Biochemical and structural studies suggest that recognition of the 5Ј-CCCTT/3Ј-GGGAA sequence triggers conformational changes in the enzyme that recruit the full set of catalytic side chains into the active site, a process that entails protein contacts with several of the nucleotide bases and specific atoms of the phosphate backbone of DNA within and immediately flanking the CCCTT element (7)(8)(9)(10)(11)(12).…”

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confidence: 99%

Nonpolar Nucleobase Analogs Illuminate Requirements for Site-specific DNA Cleavage by Vaccinia Topoisomerase

Yakovleva

Lai

Kool

et al. 2006

Journal of Biological Chemistry

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Vaccinia DNA topoisomerase forms a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate at a specific target site 5-C ؉5 C ؉4 C ؉3 T ؉2 T ؉1 p2N ؊1 in duplex DNA. Here we study the effects of nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the ؉2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the ؉1T base was not elided by F substitution. We impute a role for ؉1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the ؉4G and ؉3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for ؉4G and ؉3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the ؉1A base of the nonscissile strand; the reaction equilibrium is restored by ؉1 indole, signifying that base stacking flanking the nick is critical for the religation step. Our findings highlight base isosteres as valuable tools for the analysis of proteins that act on DNA in a site-specific manner.Vaccinia virus DNA topoisomerase IB cleaves and rejoins one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate formed at a specific pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (1). (The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Four conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265) are responsible for catalyzing the attack of the active site tyrosine (Tyr-274) on the scissile phosphodiester to form the covalent intermediate (2-6). Biochemical and structural studies suggest that recognition of the 5Ј-CCCTT/3Ј-GGGAA sequence triggers conformational changes in the enzyme that recruit the full set of catalytic side chains into the active site, a process that entails protein contacts with several of the nucleotide bases and specific atoms of the phosphate backbone of DNA within and immediately flanking the C...

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“…A paper describing the open state of the topoisomerase IB from Deinococcus radiodurans has recently appeared (10). The bacterial enzyme is much smaller than the human one, being constituted by a C-terminal domain of about 250 residues, comparable to the core subdomain III and C-terminal domain of the human enzyme, and by an N-terminal domain of about 90 residues that has no counterpart in the human enzyme.…”

Section: Introductionmentioning

confidence: 99%

The open state of human topoisomerase I as probed by molecular dynamics simulation

Chillemi

Bruselles

Fiorani

et al. 2007

Nucleic Acids Research

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The open state of human topoisomerase I has been probed by molecular dynamics simulation, starting from the coordinates of the closed structure of the protein complexed with DNA, after elimination of the 22-bp DNA duplex oligonucleotide. A repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum, after which an unperturbed simulation has been carried out for 10 ns. The simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains, which however move as a coherent unit, fully maintaining their secondary and tertiary structures. Despite movements between the domains as large as 80–90 Å, the catalytic pentad remains preassembled, the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7 Å. Electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center, in a conformation perfectly suited to interact with the negatively charged supercoiled DNA substrate.

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Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

Cited by 29 publications

References 63 publications

Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

Nonpolar Nucleobase Analogs Illuminate Requirements for Site-specific DNA Cleavage by Vaccinia Topoisomerase

The open state of human topoisomerase I as probed by molecular dynamics simulation

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