Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.

Shuman, Stewart; Kane, Eileen; Morham, Scott G.

doi:10.1073/pnas.86.24.9793

Cited by 68 publications

(38 citation statements)

References 34 publications

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“…As expected, replacement of the active-site tyrosine by phenylalanine inactivates the viral and cellular enzymes (15,(22)(23)(24). However, the Ser and Lys residues of this motif are dispensable for topoisomerase activity, insofar as alanine substitutions at Ser-270 and Lys-271 of the vaccinia enzyme are well tolerated (7).…”

Section: Mutagenesis Strategy and Production Of Mutant Proteins-mentioning

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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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: Mutagenesis Strategy and Production Of Mutant Proteins-mentioning

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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“…1). The cleavage transesterification reaction of vaccinia topoisomerase results in covalent attachment of the 32 P-labeled 12-mer 5Ј-pCGTGTCGCCCTTp to the enzyme via Tyr-274 (12,27). The unlabeled 6-mer 5Ј-OH leaving strand ATTCCC dissociates spontaneously from the protein-DNA complex.…”

Section: Effects Of Nonpolar Pyrimidine Isosteres In the Scissilementioning

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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“…The T1 residue (designated position + 1) is linked via a 3' phosphodiester bond to Tyr-274 of the enzyme (2). Synthetic model substrates have been used to examine the molecular interactions that contribute to specific cleavage at the CCCTT motif (3)(4)(5)(6)(7)(8)(9).…”

Section: Introductionmentioning

confidence: 99%

“…To exclude the contributions of covalent binding to site affinity, we focus on the DNA binding properties of Topo(Phe-274), an active site mutant protein which is unable to cleave DNA, but which binds DNA noncovalently (2,4). We find that Topo(Phe-274) does not form a stable complex with minimal substrates that are readily cleaved by the wild type enzyme.…”

Section: Introductionmentioning

confidence: 99%