No abstract

Bacterial topoisomerase I is a potential target for discovery of new antibacterial compounds. Mutant topoisomerases identified by SOS induction screening demonstrated that accumulation of the DNA cleavage complex formed by type IA topoisomerases is bactericidal. Characterization of these mutants of Yersinia pestis and Escherichia coli topoisomerase I showed that DNA religation can be inhibited while maintaining DNA cleavage activity by decreasing the binding affinity of Mg(II) ions. This can be accomplished either by mutation of the TOPRIM motif involved directly in Mg(II) binding or by altering the charge distribution of the active site region. Besides being used to elucidate the key elements for the control of the cleavage-religation equilibrium, the SOS-inducing mutants of Y. pestis and E. coli topoisomerase I have also been utilized as models to study the cellular response following the accumulation of bacterial topoisomerase I cleavage complex. Bacterial topoisomerase I is required for preventing hypernegative supercoiling of DNA during transcription. It plays an important role in transcription of stress genes during bacterial stress response. Topoisomerase I targeting poisons may be particularly effective when the bacterial pathogen is responding to host defense, or in the presence of other antibiotics that induce the bacterial stress response.

DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.

The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

The acidic residues Asp-111, Asp-113, and Glu-115 of Escherichia coli DNA topoisomerase I are located near the active site Tyr-319 and are conserved in type IA topoisomerase sequences with counterparts in type IIA DNA topoisomerases. Their exact functional roles in catalysis have not been clearly defined. Mutant enzymes with two or more of these residues converted to alanines were found to have >90% loss of activity in the relaxation assay with 6 mM Mg(II) present. Mg(II) concentrations (15-20 mM) inhibitory for the wild type enzyme are needed by these double mutants for maximal relaxation activity. The triple mutant D111A/D113A/E115A had no detectable relaxation activity. Mg(II) binding to wild type enzyme resulted in an altered conformation detectable by Glu-C proteolytic digestion. This conformational change was not observed for the triple mutant or for the double mutant D111A/D113A. Direct measurement of Mg(II) bound showed the loss of 1-2 Mg(II) ions for each enzyme molecule due to the mutations. These results demonstrate a functional role for these acidic residues in the binding of Mg(II) to induce the conformational change required for the relaxation of supercoiled DNA by the enzyme.Escherichia coli DNA topoisomerase I is the best studied representative of the type IA DNA topoisomerases. This class of enzymes includes the bacterial and archeal DNA topoisomerase I and III, reverse gyrase, and yeast and mammalian topoisomerase III, with diverse roles in cellular functions (reviewed in Refs. 1 and 2). Mg(II) is required for the interconversion of DNA topological isomers catalyzed by these enzymes. Comparison of their polypeptide sequences showed that the conserved positions include the acidic residues Asp-111, 4). When the crystal structure of the 67-kDa Nterminal transesterification domain of the enzyme was published, it was noted (5) that these three acidic residues in the active site are arranged similarly to the three acidic residues known to coordinate two divalent ions in Klenow fragment (6) that are required for the nucleotidyl transfer activity (7,8). These residues are found in domain I of the 67-kDa structure (5), which is similar to the BЈ domain of the Saccharomyces cerevisiae DNA topoisomerase II structure (9). There are corresponding acidic residues that are conserved in type IIA DNA topoisomerases (9). Severe loss of DNA relaxation and cleavage activities resulted when one of these acidic triad residues in S. cerevisiae DNA topoisomerase II, Asp-530, was mutated (10). Another conserved glutamate at Glu-9 of E. coli DNA topoisomerase I and the aspartates motif DXD at Asp-111 and Asp-113 have been proposed to be conserved motifs in a catalytic domain named Toprim found in type IA and IIA topoisomerases, as well as a number of other nucleotidyl transferases and polynucleotide cleaving activities (11). However, results of site-directed mutagenesis in E. coli DNA topoisomerase I showed that conversion of a single one of these three conserved acidic residues to alanine did not abolish the relaxation...

No abstract

No abstract

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K 0 = 0.185−1.84 cm 2 •V −1 •s −1 ), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100−500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCS N 2 ) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6−73), Tuning Mix oligomers (n = 1−5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1−4), carbonic anhydrase, β clamp (n = 1−4), topoisomerase IB, bovine serum albumin (n = 1−3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein−DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1−2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 Å 2 with <0.6% relative standard deviation (RSD)).