Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: Evidence that the gene is essential for virus growth

Shuman, Stewart; Golder, Michael; Moss, Bernard

doi:10.1016/0042-6822(89)90384-x

Cited by 69 publications

(70 citation statements)

References 13 publications

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“…Topoisomerase expression was induced by infection with bacteriophage CE6 (11). Wild type and mutant topoisomerases were purified from soluble bacterial lysates * Supported by National Institutes of Health Grant GM-46330.…”

Section: Methodsmentioning

confidence: 99%

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Mutational Analysis of 39 Residues of Vaccinia DNA Topoisomerase Identifies Lys-220, Arg-223, and Asn-228 as Important for Covalent Catalysis

Cheng

Wang

Sekiguchi

et al. 1997

Journal of Biological Chemistry

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Vaccinia DNA topoisomerase, a 314-amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3 -phosphotyrosyl)-enzyme intermediate. To identify amino acids that participate in the transesterification reaction, we introduced alanine substitutions at 39 positions within a conserved 57-amino acid segment upstream of the active-site tyrosine. Purified wild type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA. The majority of mutant proteins displayed wild type topoisomerase activity. Mutant enzymes that relaxed DNA at reduced rates were subjected to kinetic analysis of the strand cleavage and religation steps under single-turnover and equilibrium conditions. For the wild type topoisomerase, the observed single-turnover cleavage rate constant (k cl ) was 0.29 s ؊1 and the cleavage-religation equilibrium constant (K cl ) was 0.22. The most dramatic mutational effects were seen with R223A; removal of the basic side chain reduced the rates of cleavage and religation by factors of 10 ؊4.3 and 10 ؊5.0 , respectively, and shifted the cleavage-religation equilibrium in favor of the covalently bound state (K cl ‫؍‬ 1). Introduction of lysine at position 223 restored the rate of cleavage to 1 ⁄10 that of the wild type enzyme. We conclude that a basic residue is essential for covalent catalysis and suggest that Arg-223 is a constituent of the active site. Modest mutational effects were observed at two other positions (Lys-220 and Asn-228), at which alanine substitutions slowed the rates of strand cleavage by 1 order of magnitude and shifted the equilibrium toward the noncovalently bound state. Arg-223 and Lys-220 are conserved in all members of the eukaryotic type I topoisomerase family; Asn-228 is conserved among the poxvirus enzymes.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 involves 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, 2). A shared structural basis for transesterification and strand passage is inferred from the considerable amino acid sequence conservation between the cellular and virus-encoded enzymes (2, 3).The 314-amino acid vaccinia virus topoisomerase enzyme is the smallest topoisomerase known and likely constitutes the minimal functional unit of a type I enzyme (4). (The cellular type I enzymes vary in size from 765 to 1019 amino acids.) Our aim is to construct a comprehensive structure-function map of the vaccinia topoisomerase by mutagenesis. After performing initial studies via random mutagenesis (5-7), we adopted the alanine-scanning approach to define the amino acid side chains important for covalent catalysis (8, 9). The advantage of this scanning technique is that elimination of an...

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Section: Methodsmentioning

confidence: 99%

“…Plasmid pA9topo (11) was the template for the first stage PCR reaction. Gene fragments with overlapping ends obtained from the first-stage reactions were paired and used as templates in the second-stage amplification.…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis of 39 Residues of Vaccinia DNA Topoisomerase Identifies Lys-220, Arg-223, and Asn-228 as Important for Covalent Catalysis

Cheng

Wang

Sekiguchi

et al. 1997

Journal of Biological Chemistry

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“…17 The commercial enzyme preparation used in our initial studies was purchased from Epicenter (Madison, WI) but had lower and inconsistent activity.…”

Section: Vaccinia Topoisomerase Imentioning

confidence: 99%

Semi-artificial Fluorescent Molecular Machine for DNA Damage Detection

Didenko¹,

Minchew²,

Shuman³

et al. 2004

Nano Lett.

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The design of artificial molecular machines is complicated because the mechanics used in macromachines is not readily adaptable for nano environments. We constructed a semi-artificial molecular device, which contains a naturally occurring molecular machine-a vaccinia virus encoded protein-linked with an artificial part. The self-assembled construct makes two fluorescently labeled detector units. It is the first sensor capable of selectively detecting different types of DNA breaks, exemplifying a practical approach to the design of molecular devices.

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“…coli and vaccinia virus DNA topoisomerase I were purified from expression clones as described previously [25,26]. S. cerevisiae topoisomerase I was a generous gift from Dr. J.C. Wang (Harvard University).…”

Section: Enzymesmentioning

confidence: 99%

“…accumulation of positive DNA supercoils E. coli MVll90 cells containing either plasmid p1940 expressing vaccinia topoisomerase I [21,26] or its derivative p1940A (with frame shift mutation in the vaccinia virus topoisomerase gene) [21] were grown with 75 ktg/ml of novobiocin for 7 h at 30°C. The growth temperature was then shifted to 42°C to further increase the expression of vaccinia virus topoisomerase I from p1940 [21].…”

Section: In Vivo Effect Of Vaccinia Virus Topoisomerase I Expression mentioning

confidence: 99%

Vaccinia virus DNA topoisomerase I preferentially removes positive supercoils from DNA

Fernandez-Beros

Tse‐Dinh

1996

FEBS Letters

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Type I DNA topoisomerases homologous to Escherichia coli topoisomerase I normally only remove negative supercoils from DNA. Topoisomerases I from various eukaryotes share sequence homology and remove both positive and negative supercoils from DNA. Here we report that vaccinia virus topoisomerase I has significant difference in substrate preference from the other homologous type I topoisomerases. Vaccinia virus topoisomerase I shows a definite preference for removal of positive supercoiis. In contrast, topoisomerase I from human, wheat germ and Saccharomyces cerevisiae has little preference between positive and negative supercoils. The vaccinia enzyme may have evolved for functions required for optimal viral growth.

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Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: Evidence that the gene is essential for virus growth

Cited by 69 publications

References 13 publications

Mutational Analysis of 39 Residues of Vaccinia DNA Topoisomerase Identifies Lys-220, Arg-223, and Asn-228 as Important for Covalent Catalysis

Mutational Analysis of 39 Residues of Vaccinia DNA Topoisomerase Identifies Lys-220, Arg-223, and Asn-228 as Important for Covalent Catalysis

Semi-artificial Fluorescent Molecular Machine for DNA Damage Detection

Vaccinia virus DNA topoisomerase I preferentially removes positive supercoils from DNA

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