Abstract
Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.
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- Caron P. R., Wang J. C. Appendix. II: Alignment of primary sequences of DNA topoisomerases. Adv Pharmacol. 1994;29B:271–297. doi: 10.1016/s1054-3589(08)61143-6. [DOI] [PubMed] [Google Scholar]
- Denu J. M., Stuckey J. A., Saper M. A., Dixon J. E. Form and function in protein dephosphorylation. Cell. 1996 Nov 1;87(3):361–364. doi: 10.1016/s0092-8674(00)81356-2. [DOI] [PubMed] [Google Scholar]
- Gao G. J., Fonda M. L. Evidence for a phosphoenzyme intermediate formed during catalysis by pyridoxal phosphatase from human erythrocytes. Arch Biochem Biophys. 1994 Aug 15;313(1):166–172. doi: 10.1006/abbi.1994.1373. [DOI] [PubMed] [Google Scholar]
- Hanai R., Wang J. C. The mechanism of sequence-specific DNA cleavage and strand transfer by phi X174 gene A* protein. J Biol Chem. 1993 Nov 15;268(32):23830–23836. [PubMed] [Google Scholar]
- Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
- Håkansson K., Doherty A. J., Shuman S., Wigley D. B. X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes. Cell. 1997 May 16;89(4):545–553. doi: 10.1016/s0092-8674(00)80236-6. [DOI] [PubMed] [Google Scholar]
- Kumble K. D., Ahn K., Kornberg A. Phosphohistidyl active sites in polyphosphate kinase of Escherichia coli. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14391–14395. doi: 10.1073/pnas.93.25.14391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moréra S., Chiadmi M., LeBras G., Lascu I., Janin J. Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium. Biochemistry. 1995 Sep 5;34(35):11062–11070. [PubMed] [Google Scholar]
- Noirot-Gros M. F., Ehrlich S. D. Change of a catalytic reaction carried out by a DNA replication protein. Science. 1996 Nov 1;274(5288):777–780. doi: 10.1126/science.274.5288.777. [DOI] [PubMed] [Google Scholar]
- Petersen B. O., Shuman S. DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate. Nucleic Acids Res. 1997 Jun 1;25(11):2091–2097. doi: 10.1093/nar/25.11.2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roth M. J., Brown D. R., Hurwitz J. Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex. J Biol Chem. 1984 Aug 25;259(16):10556–10568. [PubMed] [Google Scholar]
- Sekiguchi J., Cheng C., Shuman S. Kinetic analysis of DNA and RNA strand transfer reactions catalyzed by vaccinia topoisomerase. J Biol Chem. 1997 Jun 20;272(25):15721–15728. doi: 10.1074/jbc.272.25.15721. [DOI] [PubMed] [Google Scholar]
- Sekiguchi J., Shuman S. Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA. Nucleic Acids Res. 1994 Dec 11;22(24):5360–5365. doi: 10.1093/nar/22.24.5360. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shuman S. DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992 Apr 25;267(12):8620–8627. [PubMed] [Google Scholar]
- Shuman S., Golder M., Moss B. Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. J Biol Chem. 1988 Nov 5;263(31):16401–16407. [PubMed] [Google Scholar]
- Shuman S., Kane E. M., Morham S. G. Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9793–9797. doi: 10.1073/pnas.86.24.9793. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shuman S., Prescott J. Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. J Biol Chem. 1990 Oct 15;265(29):17826–17836. [PubMed] [Google Scholar]
- Shuman S., Schwer B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995 Aug;17(3):405–410. doi: 10.1111/j.1365-2958.1995.mmi_17030405.x. [DOI] [PubMed] [Google Scholar]
- Shuman S. Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro. J Biol Chem. 1991 Jun 15;266(17):11372–11379. [PubMed] [Google Scholar]
- Siebers A., Altendorf K. Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli. J Biol Chem. 1989 Apr 5;264(10):5831–5838. [PubMed] [Google Scholar]
- Stivers J. T., Shuman S., Mildvan A. S. Vaccinia DNA topoisomerase I: kinetic evidence for general acid-base catalysis and a conformational step. Biochemistry. 1994 Dec 27;33(51):15449–15458. doi: 10.1021/bi00255a027. [DOI] [PubMed] [Google Scholar]
- Stivers J. T., Shuman S., Mildvan A. S. Vaccinia DNA topoisomerase I: single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions. Biochemistry. 1994 Jan 11;33(1):327–339. doi: 10.1021/bi00167a043. [DOI] [PubMed] [Google Scholar]
- Wedekind J. E., Frey P. A., Rayment I. The structure of nucleotidylated histidine-166 of galactose-1-phosphate uridylyltransferase provides insight into phosphoryl group transfer. Biochemistry. 1996 Sep 10;35(36):11560–11569. doi: 10.1021/bi9612677. [DOI] [PubMed] [Google Scholar]
- Wittschieben J., Shuman S. 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 Aug 1;25(15):3001–3008. doi: 10.1093/nar/25.15.3001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wittschieben J., Shuman S. Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis. J Biol Chem. 1994 Nov 25;269(47):29978–29983. [PubMed] [Google Scholar]
- Yokochi T., Kato J., Ikeda H. DNA nicking by Escherichia coli topoisomerase IV with a substitution mutation from tyrosine to histidine at the active site. Genes Cells. 1996 Dec;1(12):1069–1075. doi: 10.1046/j.1365-2443.1996.d01-226.x. [DOI] [PubMed] [Google Scholar]