Soluble expression, purification and characterization of the full length IS2 Transposase

<p>Abstract</p> <p>Background</p> <p>The two-step transposition pathway of insertion sequences of the IS<it>3 </it>family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS<it>2</it>, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes <it>in vitro </it>with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein.</p> <p>Results</p> <p>A soluble and active IS<it>2 </it>transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in <it>in vivo </it>transposition assays. <it>In vitro </it>electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS<it>3 </it>family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS<it>2</it>. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily.</p> <p>Conclusions</p> <p>Intractably insoluble transposases, such as the IS<it>2 </it>transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level.</p

Protein-DNA interactions define the mechanistic aspects of circle formation and insertion reactions in IS2 transposition

<p>Abstract</p> <p>Background</p> <p>Transposition in IS<it>3</it>, IS<it>30</it>, IS<it>21 </it>and IS<it>256 </it>insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS<it>2 </it>transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.</p> <p>Results</p> <p>In IS2, Step I reactions are physically and chemically asymmetric; the left imperfect, inverted repeat (IRL), the exclusive recipient end, lacks donor function. In SC I, different protection patterns of the cleavage domains (CDs) of the right imperfect inverted repeat (IRR; extensive <it>in cis</it>) and IRL (selective <it>in trans</it>) at the single active cognate IRR catalytic center (CC) are related to their donor and recipient functions. In SC II, extensive binding of the IRL CD <it>in trans </it>and of the abutted IRR CD <it>in cis </it>at this CC represents the first phase of the complex. An MCJ substrate precleaved at the 3' end of IRR revealed a temporary transition state with the IRL CD disengaged from the protein. We propose that in SC II, sequential 3' cleavages at the bound abutted CDs trigger a conformational change, allowing the IRL CD to complex to its cognate CC, producing the second phase. Corroborating data from enhanced residues and curvature propensity plots suggest that CD to CD interactions in SC I and SC II require IRL to assume a bent structure, to facilitate binding <it>in trans</it>.</p> <p>Conclusions</p> <p>Different transpososomes are assembled in each step of the IS<it>2 </it>transposition pathway. Recipient versus donor end functions of the IRL CD in SC I and SC II and the conformational change in SC II that produces the phase needed for symmetrical IRL and IRR donor attacks on target DNA highlight the differences.</p

Hot atom labeling of myoglobin and hemoglobin and biophysical studies of oxygen and carbon monoxide binding to carp hemoglobin

Human Hb, the monomeric Hb of Glycera dibranchiata and horse Mb were modified by replacement of the protoheme with 2,4-dibromodeuteroheme. Following neutron capture by \sp{79}Br and \sp{81}Br, the locations of radioactive Br were determined. Although human Hb had approximately four times the mass and volume of the other proteins, about 9% of the activated Br was inserted into each of the three globins. These results suggest that the insertion is short-range (within 15 A) and that this method could be used to label target sites in various proteins and other biological structures. Carp Hb\u27s containing proto-, meso-, deutero- and dibromoheme were prepared. Kinetic and thermodynamic parameters for oxygen and CO binding were determined at Ph 6 (+IHP) (T-state, low-affinity protein) and Ph 9 (R-state, high-affinity protein). Parameters for the binding of oxygen and CO were related to the properties of the four hemes to estimate the inductive and steric factors in the ligation process. Our results suggest that the steric factors are more important for the T-state than for the R-state. The T-state carp Hbs were very readily oxidized. Two new procedures were developed for the rapid determination of oxygen equilibrium isotherms for the T-state carp Hbs. The kinetic and thermodynamic parameters for ligation of oxygen and CO with the isolated carp $\alpha$-chains were determined. Carp $\alpha$-chains are the only hemoglobin chains isolated to date that can be classified as T-state. The secondary thermodynamic parameter ($\Delta$H\sp\circ) was found to be essential for classifying hemoglobins as T- or R-state

Thermophilic bacterial DNA polymerases with reverse-transcriptase activity

Conserved motifs found in known bacterial polI DNA polymerase sequences were identified, and degenerate PCR primers were designed for PCR amplification of an internal portion of polI genes from all bacterial divisions. We describe here a method that has allowed the rapid identification and isolation of 13 polI genes from a diverse selection of thermophilic bacteria and report on the biochemical characteristics of nine of the purified recombinant enzymes. Several enzymes showed significant reverse-transcriptase activity in the presence of Mg²⁺, particularly the polymerases from Bacillus caldolyticus EA1, Caldibacillus cellovorans CompA.2, and Clostridium stercorarium.9 page(s