Identification and characterization of the orf virus type I topoisomerase

Vaccinia topoisomerase catalyzes DNA cleavage and rejoining via transesterification to pentapyrimidine recognition site 5-(C/T)CCTT2 in duplex DNA. The proposed reaction mechanism involves general-base catalysis of the attack by active site nucleophile Tyr-274 on the scissile phosphodiester and general-acid catalysis of the expulsion of the 5-deoxyribose oxygen on the leaving DNA strand. The pK a values suggest histidine and cysteine side chains as candidates for the roles of proton acceptor and donor, respectively. To test this, we replaced each of the eight histidines and two cysteines of the vaccinia topoisomerase with alanine. Single mutants C100A and C211A and a double mutant C100A-C211A were fully active in DNA relaxation, indicating that a cysteine is not the general acid. Only one histidine mutation, H265A, affected enzyme activity. The rates of DNA relaxation, single-turnover strand cleavage, and single-turnover religation by H265A were 2 orders of magnitude lower than the wild-type rates. Yet the H265A mutation did not alter the dependence of the cleavage rate on pH, indicating that His-265 is not the general base. Replacing His-265 with glutamine or asparagine slowed DNA relaxation and single-turnover cleavage to about one-third of the wild-type rate. All three mutations, H265A, H265N, and H265Q, skewed the cleavagereligation equilibrium in favor of the covalently bound state. His-265 is strictly conserved in every member of the eukaryotic type I topoisomerase family.The eukaryotic type I DNA topoisomerase family includes the nuclear type I enzymes and the topoisomerases encoded by vaccinia and other poxviruses. These proteins relax supercoiled DNA via a common reaction mechanism, which entails noncovalent binding of the topoisomerase to duplex DNA, cleavage of one DNA strand with concomitant formation of a covalent DNA-(3Ј-phosphotyrosyl)-protein intermediate, strand passage, and strand religation (1). A shared structural basis for transesterification and strand passage is inferred from the considerable amino acid sequence conservation found by alignment of the cellular and virus-encoded enzymes (1, 2). Catalytically important residues have been identified via mutational analysis of the 314-amino acid vaccinia virus topoisomerase. Three strategies have been used: (i) random mutagenesis followed by in vivo genetic selection of mutations that adversely affect enzyme activity (3, 4); (ii) site-directed mutagenesis of specific regions of the enzyme (5-7); and (iii) targeted mutagenesis of a specific class of amino acid residues irrespective of their location within the protein. The latter approach was used to identify Tyr-274 as the active site of the vaccinia enzyme, i.e. through systematic replacement of tyrosines by phenylalanines (8).Physical mapping of the active site of yeast TOP1 to Tyr-727 (9, 10), supported by mutational analysis of the yeast, human, and vaccinia enzymes (8 -12), localizes the active site tyrosines within a common sequence element, Ser-Lys-X-X-Tyr, situated near the carboxyl ter...

Section: Discussionmentioning

confidence: 99%

Histidine 265 Is Important for Covalent Catalysis by Vaccinia Topoisomerase and Is Conserved in All Eukaryotic Type I Enzymes

Petersen

Shuman

1997

“…2 For cleavage with heavy metals, 5Ј-end-labeled oligonucleotides were precipitated with ethanol and carrier salmon sperm DNA (1 g), dried, and dissolved in 30 l of distilled water. Subsequent steps were carried out in a fume hood using chemical gloves due to the extreme hazard of phenyl mercuric acetate.…”

Section: Methodsmentioning

confidence: 99%

DNA Contacts Stimulate Catalysis by a Poxvirus Topoisomerase

Hwang

Burgin²,

Bushman

1999

Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5-(T/C)CCTT-3 sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5-(T/C)CCTT-3 sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.Topoisomerases play important roles in DNA replication, recombination, and transcription (1). Type 1B topoisomerases act ( Fig. 1) by first binding double-stranded DNA and then cleaving substrate DNA using a conserved enzyme tyrosine residue, forming a 3Ј-O-phosphotyrosine linkage. The DNA strands then isomerize, either by passing one strand through the break in the other or by rotation of the covalent complex, relaxing the DNA. The broken strand is then resealed by nucleophillic attack of the 5Ј-hydroxyl on the phosphotyrosine bond. Last, the topoisomerase dissociates from substrate.All the poxviruses studied to date encode a type 1B topoisomerase (2-7) that is inferred to be required for viral replication (8). Poxvirus topoisomerases bind to the sequence 5Ј-(T/ C)CCTT-3Ј and cleave just 3Ј of the last T (designated position ϩ1), to form the covalent intermediate (7,9,10). Although many topoisomerases have been found to show some DNA sequence preferences (11)(12)(13)(14), the high degree of specificity seen with the poxvirus topoisomerases is unique.In the case of vaccinia virus, the most thoroughly studied model, the topoisomerase enzyme consists of two domains linked through a trypsin-sensitive bridge (15,16). The N-terminal domain (amino acids 1-80) contacts the 5Ј-(T/C)CCTT-3Ј element on the major groove side of the DNA (15-17). Chemical footprinting and interference studies indicate that the enzyme contacts several phosphates, including the scissile phosphate, on both strands and the minor groove side as well. Recently, the crystal structure of the C-terminal catalytic domain of vaccinia topoisomerase (amino acids 81-314) was det...

“…(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).…”

mentioning

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

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 ...