Stewart Shuman: Topoisomerase I Structure and Mechanism

Type IB eukaryotic DNA topoisomerases modulate the topological state of DNA by cleaving and rejoining 1 strand of the DNA duplex. Cleavage is a transesterification reaction in which the scissile NpN phosphodiester is attacked by a tyrosine of the enzyme, resulting in the formation of a DNA-(3’-phosphotyrosyl)-enzyme intermediate and the expulsion of a 5’-OH polynucleotide. During strand rejoining, the 5’-OH end attacks the covalent intermediate to form a phosphodiester bond and expel the tyrosine. There is no involvement of a divalent cation in transesterification chemistry. The lambda Int family of site-specific DNA recombinases (referred to now as the tyrosine recombinases) employs an identical mechanism to catalyze the formation and resolution of Holliday junctions.

Insights into the common catalytic mechanism of the type IB topoisomerases and tyrosine recombinases have emerged from the crystal structures of Cre recombinase and human topoisomerase I bound to DNA, the unliganded structures of XerD, lambda Int, HP1 Int, and vaccinia topoisomerase, and the extensive mutational analysis of vaccinia topoisomerase. The catalytic domain of each of these proteins adopts a common fold composed of 8 alpha helices and a 3-strand antiparallel beta sheet.

Crystal Structure of the Topoisomerase Catalytic Domain

Overall fold of the topoisomerase catalytic domain. Enlarge Image Overall fold of the topoisomerase catalytic domain

We crystallized the catalytic domain of vaccinia topoisomerase and, in collaboration with our SKI colleague Nikola Pavletich, determined its structure at 2.3 Å resolution. The catalytic domain is an oblong globular protein consisting of 10 alpha helices and a 3-strand antiparallel beta-sheet on one surface. An N-terminal lobe (colored purple) comprises helices 1 to 5 and the beta-sheet; a C-terminal lobe (colored cyan) consists of helices 6 to 10. The long N-terminal alpha helix is integrated into both lobes. There was no interpretable electron density from residues 129 to 137. This segment, which is depicted as a dashed purple line connecting helices 2 and 3, includes residues that are protease-sensitive in the free topoisomerase, but protected upon DNA binding. Thus, the proteolysis and crystallographic findings together indicate that this segment is flexible in the free enzyme.

Two sulfate ions (colored red) were bound to the enzyme, which had been crystallized in ammonium sulfate. The distance between the sulfate densities was equal to the distance between vicinal backbone phosphates in 1 strand of duplex DNA. One sulfate is within hydrogen bonding distance of essential residue His-265, and essential residue Arg-223 is close by. We posit that this sulfate is located at a site that would be occupied by the scissile phosphate of the DNA substrate. Remarkably, the active site nucleophile Tyr-274 (located in helix 8) is actually oriented away from the putative binding pocket for the scissile phosphate and therefore is not poised to engage in catalysis. This implies that Tyr-274 must be reoriented upon binding of the topoisomerase to the DNA target site.

A Model of the Topoisomerase-DNA Complex

Topo I forms a C-shaped clamp around DNA. Enlarge Image Topo I forms a C-shaped clamp around DNA.

We built a model of the noncovalent topoisomerase-DNA complex by fitting the structure of the catalytic domain onto a B-form duplex, such that the sulfates are superimposed on the scissile phosphodiester and 5’-proximal phosphodiester, respectively, of the CCCTpTpA target sequence. Placement of the N-terminal domain in the major groove as dictated by the observed UV crosslinking contacts easily connected the 2 domains. The protein spans an ~18 bp DNA segment (from +10 to -9 relative to the scissile phosphodiester), which is consistent with the dimensions of the DNase I and exo III footprints. The model suggests that topoisomerase engages the DNA circumferentially, with the protein in the form of a C-shaped clamp, essentially as predicted by our footprinting studies of the topoisomerase-DNA interface. It implies conformation flexibility in the free topoisomerase, which must open up to accommodate DNA within the cleft of protein. This is most likely achieved through domain motion about the interdomain bridge segment. Also, the model places essential catalytic residues Arg-223 and His-265 in a position to interact directly with the scissile phosphodiester.

Catalytic Mechanism of Topoisomerase I

A constellation of 4 conserved amino acid side chains (e.g., Arg130, Lys167, Arg223, and His265 in the vaccinia topoisomerase) provides most of the catalytic power for the estimated 12 order-of-magnitude rate enhancement of transesterification by topoisomerase IB. The 2 arginines and the histidine interact directly with the nonbridging scissile phosphate oxygens in the DNA cocrystal structures of recombinase and human topoisomerase I. Mutational and structural data together suggest that such interactions activate the scissile phosphodiester for attack by the 5’-OH DNA or tyrosine nucleophiles; and that they enhance catalysis by stabilizing the developing negative charge on the presumptive pentacoordinate phosphorane transition state. The stereochemical outcome of the net cleavage-rejoining reaction of vaccinia topoisomerase on phosphorothioate-modified DNA is a retention of configuration at the scissile phosphodiester, as expected for 2 serial in-line SN2-type displacements.

A mechanism of general acid-base catalysis has also been proposed; whereby a general base on the enzyme (-B:) accepts a proton from the attacking tyrosine nucleophile during covalent adduct formation, and a separate enzymic general acid (-AH) donates a proton to expel the 5’-OH leaving group. In the religation step, -BH would donate a proton to the leaving tyrosine and -A: would accept a proton from the attacking 5’-OH. The amino acid side chain corresponding to -B: has not yet been identified, although many candidate residues, including all histidines, have been ruled out by mutational analysis of vaccinia topoisomerase. The available crystal structures of vaccinia and human type IB topoisomerases provide no clues to the identity of potential base catalysts that can be reconciled with mutational data; i.e., there is no essential residue in the immediate vicinity of the tyrosine nucleophile that could serve as a proton acceptor and no essential residue in direct contact with the 5’-bridging oxygen of the DNA leaving strand that could serve as the proton donor.

This situation raises the prospect that either: (i) transesterification by topoisomerase IB and tyrosine recombinases does not entail general acid-base catalysis, or (ii) the side chains on the enzyme that serve as general acid or general base are only transiently in contact with their atomic targets and their function is coordinated with conformational changes of the enzyme bound to DNA. The delineation of all relevant catalytic moieties and the conformational changes that drive transesterification is the principal challenge in the topoisomerase/tyrosine recombinase field.

Lys167 is the General Acid Catalyst of DNA Cleavage

The Enlarge Image The “general acid loop” of vaccinia topoisomerase is colored red and denoted by the arrow.

We used 5’-bridging phosphorothiolate-modified DNAs to identify Lys167 as the long-sought but elusive general acid catalyst of DNA cleavage. The lower pKa of the 5’-S leaving group versus 5’-O restored activity to the catalytically defective K167A mutant; whereas there was no positive 5’-bridging thio effect for alanine mutants of catalytic residues Arg223 and His265 that are postulated to stabilize the transition state. Lys167 is located in the loop connecting the second and third antiparallel beta strands of the catalytic domain of vaccinia topoisomerase. Lysines occupy equivalent positions in the structures of human topoisomerase I and the tyrosine recombinases. The hairpin loop, in which the lysine general acid is situated, displays considerable conformational flexibility in the various tyrosine recombinase and topoisomerase IB structures. The lysine shifts into the minor groove upon binding to the DNA target site, where it is poised to interact transiently with the 5’-O leaving group.

Cheng C, Shuman S. (2000) DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3’ mononucleotide overhang. Nucleic Acids Res. 2000;28:1893-1898.

Wittschieben J, Shuman S. (1997) Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136, and Lys-167. Nucleic Acids Res. 1997;25:3001-3008.

Wang LK, Wittschieben J, Shuman S. (1997) Mutational analysis of 26 residues of vaccinia DNA topoisomerase identifies Ser-204 as important for DNA binding and cleavage. Biochemistry. 1997;36:7944-7950.

Sekiguchi J, Shuman S. (1997) Site-specific ribonuclease activity of eukaryotic topoisomerase I. Mol Cell. 1997;1:89-97.

Wittschieben J, Petersen BO, Shuman S. (1998) Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine, or histidine converts the enzyme into a site-specific endonuclease. Nucleic Acids Res. 1998;26:490-496.

Cheng C, Kussie P, Pavletich N, Shuman S. (1998) Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases. Cell. 1998;92:841-850.

Shuman S. (1998) Polynucleotide ligase activity of eukaryotic topoisomerase I. Mol. 1998;1:741-748.

Cheng C, Shuman S. (1998) A catalytic domain of eukaryotic DNA topoisomerase I. J Biol Chem. 1998;273:11589-11595.

Krogh BO, Cheng C, Burgin A Jr, Shuman S. (1999) Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5’-bridging phosphorothiolates. Virology. 1999;264:441-451.

Krogh BO, Shuman S. (2002) Proton relay mechanism of general acid catalysis by DNA topoisomerase IB. J Biol Chem. 2002;27:5711-5714.

Krogh BO, Shuman S. (2000) DNA strand transfer catalyzed by vaccinia topoisomerase: peroxidolysis and hydroxylaminolysis of the covalent protein-DNA intermediate. Biochemistry. 2000;39:6422-6432.

Sekiguchi J, Cheng C, Shuman S. (1997) Kinetic analysis of DNA and RNA strand transfer reactions catalyzed by vaccinia topoisomerase. J Biol Chem. 1997;272:15721-15728.

Stivers JT, Jagadeesh GJ, Nawrot B, Stec WJ, Shuman S. (2000) Stereochemical outcome and kinetic effects of Rp and Sp phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase. Biochemistry. 2000;39:5561-5572.

Klemm M, Cheng C, Cassell G, Shuman S, Segall AM. (2000) Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases. J Mol Biol. 2000;299:1203-1216.

Krogh BO, Shuman S. (2000) Catalytic mechanism of DNA topoisomerase IB. Mol Cell. 2000;5:1035-1041.

Sekiguchi J, Cheng C, Shuman S. (2000) Resolution of a Holliday junction by vaccinia topoisomerase requires a spacer DNA segment 3’ of the CCCTTØ cleavage sites. Nucleic Acids Res. 2000;28:2658-2663.

Woodfield G, Cheng C, Shuman S, Burgin AB. (2000) Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3’-para-nitrophenyl phosphate ester. Nucleic Acids Res. 2000;28:3323-3331.

Cheng C, Shuman S. (2000) A recombinogenic flap ligation pathway for intrinsic repair of topoisomerase IB-induced double strand breaks. Mol Cell Biol. 2000;20:8059-8068.

Krogh BO, Claeboe CD, Hecht SM, Shuman S. (2001) Effect of 2’-5’ phosphodiesters on DNA transesterification by vaccinia topoisomerase. J Biol Chem. 2001;276:20907-20912.

Krogh BO, Shuman S. (2001) Vaccinia topoisomerase mutants illuminate conformational changes during closure of the protein clamp and assembly of a functional active site. J Biol Chem. 2001;276:36091-36099.

Krogh BO, Shuman S. (2002) A poxvirus-like type IB topoisomerase family in bacteria. Proc Natl Acad Sci USA. 2002;99:1853-1858.

Cheng C, Shuman S. (1999) Site-specific transesterification by vaccinia topoisomerase: role of specific phosphates and nucleosides. Biochemistry. 1999;38:16599-165612.

Sekiguchi J, Stivers JT, Mildvan AS, Shuman S. (1996) Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin. J Biol Chem. 1996;271:2313-2322.

Shuman S. (1992) DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992;267:8620-8627.

Shuman S. (1992) Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992;267:16755-16758.

Stivers JT, Shuman S, Mildvan AS. (1994) Vaccinia DNA topoisomerase I: single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions. Biochemistry. 1994;33:327-339.

Wittschieben J, Shuman S. (1994) Mutational analysis of vaccinia virus DNA topoisomerase defines amino acid residues essential for covalent catalysis. J Biol Chem. 1994;269:29978-29983.

Sekiguchi J, Shuman S. (1994) Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA. Nucleic Acids Res. 1994;22:5360-5365.

Sekiguchi J, Shuman S. (1994) Stimulation of vaccinia DNA topoisomerase by nucleoside triphosphates. J Biol Chem. 1994;269:29760-29764.

Stivers JT, Shuman S, Mildvan AS. (1994) Vaccinia DNA topoisomerase I: kinetic evidence for general acid-base catalysis and a conformational step. Biochemistry. 1994;33:15449-15458.

Sekiguchi J, Shuman S. (1994) Vaccinia topoisomerase binds circumferentially to DNA. J Biol Chem. 1994;269:31731-31734.

Shuman S. (1994) Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem. 1994;269:32678-32684.

Shuman S, Bear DG, Sekiguchi J. (1997) Intramolecular synapsis of duplex DNA by vaccinia topoisomerase. EMBO J. 1997;16:6584-6589.

Petersen BO, Wittschieben J, Shuman S. (1996) Mutations within a conserved region of vaccinia topoisomerase affect the cleavage-religation equilibrium. J Mol Biol. 1996;263:181-195.

Sekiguchi J, Shuman S. (1997) Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding. Nucleic Acids Res. 1997;25:3649-3656.

Sekiguchi J, Seeman NC, Shuman S. (1996) Resolution of Holliday junctions by eukaryotic DNA topoisomerase I. Proc Natl Acad Sci USA. 1996;93:785-789.

Sekiguchi J, Shuman S. (1996) Identification of contacts between topoisomerase I and its target DNA by site-specific photocrosslinking. EMBO J. 1996;15:3448-3457.

Sekiguchi J, Shuman S. (1996) Covalent DNA binding by vaccinia topoisomerase results in unpairing of the thymine base 5’ of the scissile bond. J Biol Chem. 1996;271:19436-19442.

Petersen BO, Hall RL, Moyer RW, Shuman S. (1997) Characterization of a DNA topoisomerase encoded by Amsacta moorei entomopoxvirus. Virology. 1997;230:197-206.

Petersen BO, Shuman S. (1997) Histidine-265 is important for covalent catalysis by vaccinia topoisomerase and is conserved in all eukaryotic type I enzymes. J Biol Chem. 1997;272:3891-3896.

Petersen BO, Shuman S. (1997) DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate. Nucleic Acids Res. 1997;25:2091-2097.

Wang LK, Shuman S. (1997) Deletions at the carboxyl terminus of vaccinia DNA topoisomerase affect DNA binding and enhance distributivity in DNA relaxation. Biochemistry. 1997;36:3909-3916.

Cheng C, Wang LK, Sekiguchi J, Shuman S. (1997) Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis. J Biol Chem. 1997;272:8263-8269.

Shuman S. (1991) Recombination mediated by vaccinia DNA topoisomerase I in Escherichia coli is sequence specific. Proc Natl Acad Sci USA. 1991;88:10104-10108.

Sekiguchi J, Shuman S. (1995) Proteolytic footprinting of vaccinia topoisomerase I bound to DNA. J Biol Chem. 1995;270:11636-11645.