Melanoplus sanguinipes Entomopoxvirus DNA Topoisomerase: Site-Specific DNA Transesterification and Effects of 5′-Bridging Phosphorothiolates

Krogh, Berit Olsen; Burgin, Alex B.; Shuman, Stewart

doi:10.1006/viro.1999.0022

Cited by 18 publications

(18 citation statements)

References 39 publications

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“…1A). An explanation for the observed altered equilibrium is that the 5Ј-sulfhydryl leaving strand in the cleavage reaction is an extremely poor nucleophile in the religation step (16,17). The reaction rates of wild-type topoisomerase on the 5Ј-O and 5Ј-S equilibrium substrates were identical within our limits of detection; both reactions were effectively complete in 5 s (the earliest time examined).…”

Section: Involvement Of Arg-130 In General Acidmentioning

confidence: 71%

Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB

Krogh

Shuman

2002

Journal of Biological Chemistry

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Type IB topoisomerases cleave and rejoin DNA through a DNA-(3-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes the attack of the tyrosine nucleophile (Tyr-274) at the scissile phosphodiester. Previous studies implicated Arg-223 and His-265 in transition state stabilization and Lys-167 in proton donation to the 5-O of the leaving DNA strand. Here we find that Arg-130 also plays a major role in leaving group expulsion. The rate of DNA cleavage by vaccinia topoisomerase mutant R130K, which was slower than wild-type topoisomerase by a factor of 10 ؊4.3 , was stimulated 2600-fold by a 5-bridging phosphorothiolate at the cleavage site. The catalytic defect of the R130A mutant was also rescued by the 5-S modification (190-fold stimulation), albeit to a lesser degree than R130K. We surmise that Arg-130 plays dual roles in transition state stabilization and general acid catalysis. Whereas the R130A mutation abolishes both functions, R130K permits the transition state stabilization function (via contact of lysine with the scissile phosphate) but not the proton transfer function. Our results show that the process of general acid catalysis is complex and suggest that Lys-167 and Arg-130 comprise a proton relay from the topoisomerase to the 5-O of the leaving DNA strand.Type IB DNA topoisomerases relax DNA supercoils via a 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 (1, 2). Tyrosine recombinases use a similar transesterification mechanism to form and resolve Holliday junctions. The catalytic domains of topo 1 IB and tyrosine recombinases adopt a common fold composed of eight ␣ helices and a three-stranded antiparallel ␤ sheet (3-9). The constituents of the active site occupy similar positions in the topo IB and recombinase tertiary structures.Four conserved amino acid side chains (e.g. Arg-130, Lys-167, Arg-223, and His-265 in the vaccinia topoisomerase) catalyze the attack of the active site tyrosine nucleophile (Tyr-274) on the scissile phosphodiester (10 -12). Mutational, stereochemical, and structural data for vaccinia and nuclear topoisomerase IB and tyrosine recombinases suggest that the two arginines and the histidine contact the nonbridging oxygens of the scissile phosphodiester and that these interactions serve to stabilize a proposed pentacoordinate phosphorane transition state (3, 5, 9, 10 -13).Recently we used 5Ј-bridging phosphorothiolate-modified DNAs to implicate Lys-167 of vaccinia topoisomerase as a general acid catalyst of the DNA cleavage reaction (14). The hypothesis was that if the expulsion of the 5Ј-oxygen of the leaving DNA strand was indeed catalyzed by a general acid on the topoisomerase, then the requirement for the general acid ought to be alleviated by introducing a 5Ј-bridging phosphorothiolate at the scissile phosphodiest...

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Section: Involvement Of Arg-130 In General Acidmentioning

confidence: 71%

Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB

Krogh

Shuman

2002

Journal of Biological Chemistry

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“…(The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (2)(3)(4)(5)(6), despite the large variations in overall G/C contents of the genomes of the different poxvirus genera. Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the 5Ј-CCCTT/3Ј-GGGAA target sequence (7,8).…”

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confidence: 99%

“…Position-specific interference by methylphosphonate modifications at remote phosphates on the scissile and nonscissile strands has provided an atomic resolution map of the DNA backbone contacts required for active site assembly (8). Whereas sterically subtle modifications of nonbridging phosphate oxygens flanking the cleavage site can have drastic effects on transesterification chemistry (8), phosphorothiolate substitutions for the 5Ј-bridging oxygens of the scissile strand have no significant effect on the rate of DNA cleavage by vaccinia topoisomerase (6).…”

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confidence: 99%

Individual Nucleotide Bases, Not Base Pairs, Are Critical for Triggering Site-specific DNA Cleavage by Vaccinia Topoisomerase

Tian

Sayer

Jerina

et al. 2004

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 abasic lesions at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. The rate of DNA incision was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the ؊1N, ؉1T, ؉2T, and ؉4C bases of the scissile strand, but abasic lesions at ؉5C and ؉3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of ؉4G, ؉3G, ؉2A, and ؉1A reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at ؉5G and ؊1N had no effect. The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. The rate of single-turnover DNA religation by the covalent topoisomerase-DNA complex was insensitive to abasic sites within the CCCTT sequence of the scissile strand, but an abasic lesion at the 5-OH nucleoside (؊1N) of the attacking DNA strand slowed the rate of religation by a factor of 600. Nonscissile strand abasic lesions at ؉1A and ؊1N slowed the rate of religation by factors of ϳ140 and 20, respectively, and strongly skewed the cleavage-religation equilibrium toward the covalent complex. Thus, abasic lesions immediately flanking the cleavage site act as topoisomerase poisons.Poxvirus topoisomerases are exemplary type IB topoisomerase family members; they cleave and rejoin one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate. Vaccinia topoisomerase cleaves duplex DNA at a pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (1). (The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (2-6), despite the large variations in overall G/C contents of the genomes of the different poxvirus genera. Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the 5Ј-CCCTT/3Ј-GGGAA target sequence (7,8).Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV cross-linking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (9 -15). Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification. For example, positionspecific covalent polycyclic aromatic hydrocarbon diol epoxide-DNA adducts have been exploited to probe the minor groove interface (16) and the effects of intercalation at all of the dinucleotides steps spanning the target site (17, 18). The aromatic hydrocarbon adduct studies delineated the ...

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“…Introduction of a single 5Ј-bridging phosphorothiolate linkage (C3Ј-O-[PO 2 ]-S-C5Ј) at the scissile phosphodiester of an equilibrium duplex substrate results in the trapping of mammalian and poxvirus TopIB in the covalently bound state (3,16,18). An explanation for the altered equilibrium is that the 5Ј-SH leaving strand in the cleavage reaction is a relatively poor nucleophile in the religation step.…”

Section: Resultsmentioning

confidence: 99%

“…A notable finding was that MimiTopIB is able to cleave DNA at the same 5Ј-CCCTT2 site recognized by all poxvirus DNA topoisomerases (13,14,18,26,28,33). We determined the kinetic and equilibrium parameters for transesterification by MimiTopIB at this site.…”

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

Benarroch

Claverie

Raoult

et al. 2006

J Virol

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Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes dozens of proteins with imputed functions in nucleic acid transactions. Here we produced, purified, and characterized mimivirus DNA topoisomerase IB (TopIB), which we find to be a structural and functional homolog of poxvirus TopIB and the poxvirus-like topoisomerases discovered recently in bacteria. Arginine, histidine, and tyrosine side chains responsible for TopIB transesterification are conserved and essential in mimivirus TopIB. Moreover, mimivirus TopIB is capable of incising duplex DNA at the 5-CCCTT cleavage site recognized by all poxvirus topoisomerases. Based on the available data, mimivirus TopIB appears functionally more akin to poxvirus TopIB than bacterial TopIB, despite its greater primary structure similarity to the bacterial TopIB group. We speculate that the ancestral bacterial/viral TopIB was disseminated by horizontal gene transfer within amoebae, which are permissive hosts for either intracellular growth or persistence of many present-day bacterial species that have a type IB topoisomerase.DNA topoisomerases are found in all free-living organisms; they modify DNA topology by introducing reversible enzymelinked breaks in the DNA phosphodiester backbone (reviewed in reference 6). Topoisomerases accomplish this either by cleaving one strand of the DNA duplex and passing the intact complementary strand through the nick (type I topoisomerase) or by cleaving both strands and passing an intact duplex segment through the double-strand break (type II topoisomerase). Type II topoisomerases are oligomeric proteins that form covalent tyrosyl-5Ј-phosphodiester linkages at the two sites of strand scission. Type I enzymes are monomeric and are classified as having either type IA or type IB mechanisms. Type IA enzymes form a tyrosyl-5Ј-phosphodiester linkage at the break site, whereas type IB topoisomerases generate a tyrosyl-

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Melanoplus sanguinipes Entomopoxvirus DNA Topoisomerase: Site-Specific DNA Transesterification and Effects of 5′-Bridging Phosphorothiolates

Cited by 18 publications

References 39 publications

Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB

Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB

Individual Nucleotide Bases, Not Base Pairs, Are Critical for Triggering Site-specific DNA Cleavage by Vaccinia Topoisomerase

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

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