Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease

112

Protein primers are used to initiate genomic synthesis of several RNA and DNA viruses, although the structural details of the primer-polymerase interactions are not yet known. Poliovirus polymerase binds with high affinity to the membrane-bound viral protein 3AB but uridylylates only the smaller peptide 3B in vitro. Mutational analysis of the polymerase identified four surface residues on the three-dimensional structure of poliovirus polymerase whose wild-type identity is required for 3AB binding. These mutants also decreased 3B uridylylation, arguing that the binding sites for the membrane tether and the protein primer overlap. Mutation of flanking residues between the 3AB binding site and the polymerase active site specifically decreased 3B uridylylation, likely affecting steps subsequent to binding. The physical overlap of sites for protein priming and membrane association should facilitate replication initiation in the membrane-associated complex.The replication of linear nucleic acids without loss of coding information from the ends is a problem that has been solved in several ways by cellular and viral genomes. Solutions include those used by vaccinia virus, which primes DNA replication from hairpins that can be refolded to regenerate the terminal sequences (1, 2), by Drosophila, which repair damaged chromosomal termini by recombination (3), by many eukaryotic cells, which add end-specific telomeric sequences postreplicatively, and by viruses such as adenovirus and ⌽29, which use protein primers that are covalently linked to the initiating nucleotides (reviewed in Ref. 4). For mammalian positive-strand RNA viruses such as poliovirus and hepatitis C, two mechanisms are suspected: de novo initiation (5-8) and protein priming (9) from the genomic ends.The protein primer for the synthesis of poliovirus RNA includes, at a minimum, the 22-amino acid viral peptide 3B (also called VPg), which is found covalently linked to the 5Ј ends of all newly synthesized positive and negative strands. Poliovirus translates its proteins as a single, large polyprotein that is cleaved into the proteins required for virion formation, host modification, and RNA replication. In many cases, proteolytic precursors have functions distinct from those of the limit digestion products. Evidence for the use of 3B. As a protein primer comes from in vitro experiments in which it was demonstrated that 3B is uridylylated in the presence of UTP, the poliovirus RNA-dependent RNA polymerase (3D), and an RNA template (9). The RNA used to template the uridylylation of 3B can either be poly(A), poliovirus RNA, or the small cis replication enhancer RNA (10, 11), an internal sequence required for RNA replication in infected cells (12). Whether 3B itself serves as the primer within infected cells and how it is brought into the RNA replication complex are not yet known.Evidence for direct binding between polymerase and 3B was observed in the two-hybrid system (13), although a stronger signal was observed between the polymerase and a larger polypeptide th...

mentioning

confidence: 56%

Similar Structural Basis for Membrane Localization and Protein Priming by an RNA-dependent RNA Polymerase

Lyle

Clewell

Richmond

et al. 2002

112

“…In addition to its principal activity of DNA relaxation, the reactions catalyzed by the vaccinia topoisomerase I include resolution of Holliday junctions and the production of a 2Ј,3Ј-cyclic phosphate by a mechanism analogous to Flp RNase I (19,35). The topoisomerase can mediate a strand joining reaction in which the 5Ј-hydroxyl end of a DNA chain attacks the cyclic phosphate (36) and can be made to act as an endonuclease by replacing its active site tyrosine with glutamic acid (37). The altered reactivity is reminiscent of the conversion of the endonuclease NaeI into a topoisomerase or of E. coli topoisomerase IV into an endonuclease by single amino acid changes in each instance (38,39).…”

Section: Competition Between the Rna Cleavage Activities Of Flp And Tmentioning

confidence: 99%

Flp Ribonuclease Activities

Ahn

Pathania

et al. 1998

The ribonuclease active site harbored by the Flp sitespecific recombinase can act on two neighboring phosphodiester bonds to yield mechanistically distinct chain breakage reactions. One of the RNase reactions apparently proceeds via a covalent enzyme intermediate and targets the phosphodiester position involved in DNA recombination (Flp RNase I activity). The second activity (Flp RNase II) targets the phosphodiester immediately to the 3 side but appears not to involve an enzymelinked intermediate. Flp RNase I is absolutely dependent upon Tyr-343 of Flp and is competitive with respect to the normal strand joining reaction. It can utilize the 2-hydroxyl group from any one of the four ribonucleotides with comparable efficiencies in the cleavage reaction. On the other hand, the RNase II reaction mediated by Flp(Y343F) is specific for U and cannot utilize the 2-hydroxyl group from ribo-A, -G, or -C under standard reaction conditions. The RNase II activity is also sensitive to the 3-neighboring base. Although dT is functional, the activity is stimulated by U or U-2-OMe. The Flp RNase II reaction effectively competes with the normal strand cleavage reaction mediated by Tyr-343, even though their phosphodiester targets are not the same.The Flp protein encoded by the 2-m plasmid of Saccharomyces cerevisiae is a site-specific DNA recombinase that is thought to play a central role in the copy number control of this extrachromosomal element (reviewed in Ref. 1). Flp is a member of the integrase family of "conservative" site-specific recombinases. Members of this family utilize a common biochemical mechanism to carry out a wide range of biological functions (2-4).The int(egrase) family recombinases harbor four invariant residues located within two domains of modest amino acid homology: an arginine-histidine-arginine triad (RHR) 1 and a tyrosine residue, corresponding to Arg-191, His-305, Arg-308, and Tyr-343 of Flp (see sequence alignments in Refs. 5-7). The RHR triad and Tyr-343 of Flp appear to be directly involved in the catalytic steps of recombination (reviewed in Ref. 8). The tyrosine residue provides the nucleophile during the DNA strand cleavage reaction. The resulting products are a 3Ј-Ophosphotyrosyl bond between Flp and the broken DNA end, and a 5Ј-hydroxyl group. Strand joining is chemically equivalent to the reverse of the cleavage reaction. The 5Ј-hydroxyl acts as the nucleophile to displace the DNA-linked tyrosine, restoring the continuity of the DNA strand. During a normal recombination event, this 5Ј-hydroxyl group originates from the cleaved strand of the partner DNA molecule.Mutations of the RHR triad residues or Tyr-343 of Flp result in the arrest of recombination at the cleavage step, or at the strand transfer step, or both (9 -12). It has been generally believed that the likely catalytic role of the RHR triad is in orienting the target, the scissile phosphodiester in the DNA chain or the 3Ј-O-phosphotyrosyl bond formed as a result of cleavage, for nucleophilic attack. The recently solved crystal structu...

“…Catalytic site residues Arg 130 , Lys 167 , and Arg 223 were also substituted with methionine in order to maintain similar hydrophobic contacts along the length of the side chain, while selectively eliminating the charged functional group, a type of substitution not previously studied. The Tyr 274 catalytic tyrosine was substituted with Phe to remove the hydroxyl group that serves as the nucleophile in the transesterification reaction mediating initial cleavage, a type of substitution also studied previously (16,29).…”

Section: Selection Of Amino Acids Formentioning

confidence: 99%

“…Previous studies have shown that the sequence-specific recognition is involved not only in initial DNA binding but in a postbinding conformational step that promotes cleavage (4,6,15). Very extensive mutagenesis of the vaccinia topoisomerase has identified residues involved in catalysis and additional residues important for activation of cleavage after DNA binding (4,6,(15)(16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

Regulation of Catalysis by the Smallpox Virus Topoisomerase

Hwang

Minkah

Perry

et al. 2006

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp 168 is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.All poxviruses encode topoisomerases of the type IB family (1, 2). These enzymes release DNA supercoils via a mechanism involving a 3Ј-phosphotyrosine covalent protein-DNA intermediate (Fig. 1A). In this reaction, the topoisomerase first binds to DNA, then catalyzes nucleophilic attack of an enzyme tyrosine residue on one DNA strand, forming a 3Ј-phosphotyrosine linkage. This resulting DNA strand break allows rotation of the continuous DNA strand around the nick, thereby releasing supercoils (3, 4). The reaction cycle is completed by religation, in which the hydroxyl group of the free DNA 5Ј-end at the nick attacks the phosphotyrosine linkage, rejoining the DNA, followed by product release (5, 6).