The Domain Organization and Properties of Individual Domains of DNA Topoisomerase V, a Type 1B Topoisomerase with DNA Repair Activities

Abstract

Topoisomerase V (Topo V) is a type IB (eukaryoticlike) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/ apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37°C and 80°C. The Topo V protein is comprised of (i) a 44-kDa NH 2 -terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH 2 -terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.Methanopyrus kandleri DNA topoisomerase V (Topo V) 1 (1-5) relaxes both negatively and positively supercoiled DNA in the temperature range from 60 to 122°C by catalyzing the transient breakage of a phosphodiester bond in a single DNA strand (reviewed in Refs. 6 -8)). Topo V is active in an unsurpassable range of monovalent salt concentrations, from 0 to 0.65 M KCl or NaCl and from 0 to 3.1 M of potassium glutamate (K-Glu), and no metal cation or energy cofactor is required for Topo V activity. Cleavage of a phosphodiester bond in DNA involves a transesterification reaction in which the nucleophilic O-4 oxygen of the active site tyrosine (amino acid 226 in Topo V) (5) attacks the phosphodiester linkage (6). This results in the formation of a phosphotyrosine bond between the enzyme and the 3Ј end of the broken strand. This covalent intermediate can be trapped by denaturing the enzyme during catalysis with either SDS or alkali (1, 9). In vitro formation of covalent Topo V-DNA complexes involving regular duplex DNA is, however, very inefficient. The one cleavage site mapped so far resembles the consensus site for DNA cleavage by eukaryotic topoisomerase I in the absence of camptothecin (1).Unlike other known DNA topoisomerases, Topo V has associated apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair (5). The protein incises the phosphodiester backbone at the AP site, and then at the AP endonuclease-cleaved AP site, it removes the 5Ј-2-deoxyribose-5-phosphate moiety so that a single-nucleotide gap with a 3Ј-hydroxyl and 5Ј-phosphate can be filled by a DNA poly...

Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

“…Topoisomerase activity is present in the Ϸ30-kDa fragment at the N-terminal part of the enzyme, whereas the rest of the protein is formed by 24 tandem helix-hairpin-helix (HhH) repeats, some of which are involved in DNA repair (4,13). Topo V is a hyperthermophilic enzyme with a temperature optimum of 108°C for its topoisomerase activity (1).…”

Biochemical Characterization of the Topoisomerase Domain of Methanopyrus kandleri Topoisomerase V

“…The pET15bT44 plasmid carries the coding sequence for the 44-kDa amino-terminal fragment of Topo-V (Topo-44) that extends to residue 380 (35). The pET14bT78 construct encodes for the 78-kDa N-terminal fragment of Topo-V (Topo-78) and spans residues 1-685 (32). For mutagenesis, DNA primers (Integrated DNA Technologies) were designed for use with the QuikChange site-directed mutagenesis kit (Stratagene).…”

“…Experiments were also done with mutants made in the Topo-78 fragment. Topo-78 has seven full (HhH) 2 domains and is a fragment with both topoisomerase and DNA repair activities (31,32). The Topo-78 backbone helped to assess the effect of mutations in instances where the corresponding Topo-44 mutant was inactive, presumably due to protein-DNA binding effects mediated by the (HhH) 2 domains.…”

“…It was first identified in the hyperthermophilic archaeon M. kandleri and was later found in other Methanopyrus species. Topo-V is a two-domain protein, with an N-terminal topoisomerase domain followed by 24 helix-hairpin-helix (HhH) motifs arranged as 12 tandem (HhH) 2 domains (8,31,32). Some of the (HhH) 2 domains are involved in DNA repair, and there are at least two apurinic/apyrimidinic site-processing active sites (31,33).…”

A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

“…Although further experiments are necessary, one possibility is that the motif works as a binding module to ␣-glucan, just like a plant BE that attaches to small newborn starch granules (31). Alternatively, the motif may be related to the stability of the protein (other than thermostability), as in the case of multiple copies of HhH motifs in Methanopyrus kandleri DNA topoisomerase V that are responsible for salt tolerance (4,30). In the structural genomics project of Thermus thermophilus HB8 (http://www.thermus.org/), the crystal structure of a GH-57-type BE ortholog, the TT1467 protein, was determined, and the data are filed in the Protein Data Bank (accession number 1UFA).…”