Biomedical subjects

U Curth

Publications and source records attributed to U Curth.

10 recordsLinked to original sources

PubMed2000

Interaction of E. coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease.

The 3'-5' single-stranded DNA(ssDNA) degrading exonuclease I of E. coli directly interacts with the E. coli ssDNA binding protein (EcoSSB). Analytical ultracentrifugation shows that all 4 carboxy-termini of an EcoSSB tetramer bind exonuclease I. Binding is weakened by increasing salt concentrations, indicating the involvement of the negatively charged amino acids of the carboxy-terminus of SSB. Mutant SSB proteins EcoSSBP176S (ssb-113) and EcoSSBF177C do not bindtoexonuclease I while EcoSSBG15D (ssb-3) does bind. In a co-precipitation assay we show that the absence of the lastten amino acids (PMDFDDDIPF) completely abolishes binding of EcoSSB to exonuclease I. The interaction does not depend on the presence of the correct amino-terminal DNA binding domain or the amino acid sequences between the DNA binding domain and the last ten amino acids. A synthetic peptide (WMDFDDDIPF), corresponding to the last nine amino acids of EcoSSB, specifically inhibits the interaction. Both EcoSSBP176S and EcoSSBF177C SSBs bind DNA similar to wild-type EcoSSB, indicating that the phenotype of ssb-113 is not an indication of altered DNA binding. The repair deficiency of either ssb-3 or ssb-113 strain can be complemented by overexpression of the respective other mutant.

Binding Sites↗

PubMed1998

Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli.

Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein to tyrosine or lysine leads to cells which are UV- and temperature-sensitive. The defects of both ssbH55Y (ssb-1) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation producing a less stable, readily dissociating protein whose more severe replication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was suppressed by spontaneous mutations that changed the glutamine at position 76 or 110 to leucine. Using guanidinium chloride denaturation monitored by sedimentation diffusion equilibrium experiments in the analytical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable than wild-type SSB. Additionally, the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denaturation than wild-type SSB protein.

Amino Acids↗

PubMed1997

A common core for binding single-stranded DNA: structural comparison of the single-stranded DNA-binding proteins (SSB) from E. coli and human mitochondria.

The crystal structure of the DNA-binding domain of E. coli SSB (EcoSSB) has been determined to a resolution of 2.5 A. This is the first reported structure of a prokaryotic SSB. The structure of the DNA-binding domain of the E. coli protein is compared to that of the human mitochondrial SSB (HsmtSSB). In spite of the relatively low sequence identity between them, the two proteins display a high degree of structural similarity. EcoSSB crystallises with two dimers in the asymmetric unit, unlike HsmtSSB which contains only a dimer. This is probably a consequence of the different polypeptide chain lengths in the EcoSSB heterotetramer. Crucial differences in the dimer-dimer interface of EcoSSB may account for the inability of EcoSSB and HsmtSSB to form cross-species heterotetramers, in contrast to many bacterial SSBs.

Crystallography, X-Ray↗

PubMed1997

Crystal structure of human mitochondrial single-stranded DNA binding protein at 2.4 A resolution.

We solved the crystal structure of the homotetrameric single-stranded DNA binding (SSB) protein from human mitochondria at a resolution of 2.4 A. The tetramer is formed by two dimers interacting head-to-head and shows D2 symmetry. Sequence-related tetrameric SSB proteins occur in prokaryotes and eukaryotic mitochondria; this is the first report of an atomic resolution structure of this type of protein. Using biochemical data and analysis of sequence homologies, we were able to correlate the functional properties with structure. We propose that ssDNA wraps around the tetrameric HsmtSSB protein through electropositive channels guided by flexible loops.

Amino Acid Sequence↗

PubMed1996

In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein.

We constructed several deletion mutants of Escherichia coli single-stranded DNA binding protein (EcoSSB) lacking different parts of the C-terminal region. This region of EcoSSB is composed of two parts: a glycine and proline-rich sequence of approximately 60 amino acids followed by an acidic region of the last 10 amino acids which is highly conserved among the bacterial SSB proteins. The single-stranded DNA binding protein of human mitochondria (HsmtSSB) lacks a region homologous to the C-terminal third of EcoSSB. Therefore, we also investigated a chimeric protein consisting of the complete sequence of the human mitochondrial single-stranded DNA binding protein (HsmtSSB) and the C-terminal third of EcoSSB. Fluorescence titrations and DNA-melting curves showed that the C-terminal third of EcoSSB is not essential for DNA-binding in vitro. The affinity for single-stranded DNA and RNA is even increased by the removal of the last 10 amino acids. Consequently, the nucleic acid binding affinity of HsmtSSB is reduced by the addition of the C-terminus of EcoSSB. All mutant proteins lacking the last 10 amino acids are unable to substitute wild-type EcoSSB in vivo. Thus, while the nucleic acid binding properties do not depend on an intact C-terminus, this region is essential for in vivo function. Although the DNA binding properties of HsmtSSB and EcoSSB are quite similar, HsmtSSB does not function in E.coli. This failure cannot be overcome by fusing the C-terminal third of EcoSSB to HsmtSSB. Thus differences in the N-terminal parts of both proteins must be responsible for this incompatibility. None of the mutants was defective in tetramerization. However, mixed tetramers could only be formed by proteins containing the same N-terminal part. This reflects structural differences between the N-terminal parts of HsmtSSB and EcoSSB. These results indicate that the region of the last 10 amino acids, which is highly conserved among bacterial SSB proteins, is involved in essential protein-protein interactions in the E.coli cell.

Bacterial Proteins↗

PubMed1995

Stability of Escherichia coli single-stranded DNA binding protein (EcoSSB)

Conformation and stability of EcoSSB, a single-stranded DNA binding protein encoded by Escherichia coli, were analyzed by circular dichroism and fluorescence measurements. From CD measurements at pH 7.5, EcoSSB can be classified as a protein with high alpha-helix and beta-sheet content. The hydrophobicity of the environment of the tryptophan residues of the native protein is only marginally increased in comparison to the unfolded protein. The GdnHCl induced unfolding curves measured by CD and fluorescence are coincident and sigmoidal and show a monophasic transition. The stability of EcoSSB is concentration dependent and the unfolding behavior can be described as a two-state transition from the folded tetrameter to unfolded monomers. The mean values of free energy of dissociation and unfolding delta GH2O mu are between 173 and 177 kJ.mol-1 and the mean half concentration c1/2 of GdnHCl of the transition curves are about 1.5 M and 1.7 M for protein concentrations of 0.1 mg.ml-1 and 0.5 mg.ml-1, respectively.

Circular Dichroism↗

PubMed1994

Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties.

The gene for the mature human mitochondrial single-stranded-DNA binding protein (HsmtSSB) has been transferred into a protein-overproducing vector and expressed in Escherichia coli. The protein was purified to homogeneity and its physicochemical properties were investigated. From sequence comparison, HsmtSSB shows some similarities to the N-terminal part of the single-stranded DNA-binding protein (SSB) from E. coli (EcoSSB). Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C-terminally shortened EcoSSB. Electron-microscopic images of the free protein show a globular tetrahedral structure. Binding of poly(desoxythymidylic acid) [poly(dT)] leads to a reduction of the tryptophan fluorescence of the protein up to 96%. Fluorescence titrations with poly(dT) show apparent binding-site sizes of 50-70 nucleotides/tetramer between 0.05 M and 2 M NaCl. Binding to poly(dT) proceeds in a nearly diffusion-controlled reaction with an association-rate constant kass of 4 x 10(8) M-1s-1. The rate-limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein on the linear matrix is fast. Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands. This leads to the conclusion that the binding cooperativity is low (omega < 150). The two tryptophans of HsmtSSB were replaced by threonine and tyrosine. The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA-binding affinity of the protein.

Binding Sites↗

PubMed1993

Multiple binding modes of the single-stranded DNA binding protein from Escherichia coli as detected by tryptophan fluorescence and site-directed mutagenesis.

We have systematically substituted the four tryptophan residues of the single-stranded DNA binding protein from Escherichia coli (EcoSSB) by polar (serine or threonine) and aromatic (tyrosine or phenylalanine) amino acids. The resulting mutants with either single amino acid exchanges or triple substitutions are all active in ssDNA binding, though in some cases with reduced affinities. Measurements of the fluorescence of the mutated EcoSSBs show that there is no interaction between the four different tryptophan residues. We analyzed the ssDNA binding of the mutant proteins by fluorescence titrations. At 0.3 M NaCl ("high salt"), all singly substituted proteins bind to poly(dT) in a manner comparable to wild-type EcoSSB, covering 65 nucleotides with 1 EcoSSB tetramer. W54S mutant protein is an exception since even at 0.3 M NaCl it covers approximately 35 nucleotides, a behavior which is typical of salt concentrations below 10 mM NaCl ("low salt"). From this observation, it is inferred that tryptophan-54 is involved in a direct interaction with the ssDNA favoring the "high-salt" binding mode. All mutant proteins lacking tryptophan-54 but possessing tryptophan-88 at "low-salt" concentrations show a nonmonotonous behavior in the fluorescence titrations. This behavior can be interpreted assuming a model of cooperative binding of EcoSSB to poly(dT) with two different binding site sizes (n approximately 27 and n approximately 33) and different binding affinities. A quantitative treatment of the problem of multiple binding modes in the interaction of a multidentate ligand with a linear polymer is applied to these titrations.

Amino Acid Sequence↗

PubMed1993

Viability and preliminary in vivo characterization of site-directed mutants of Escherichia coli single-stranded DNA-binding protein.

Site-directed mutations involving selected amino acids of Escherichia coli single-stranded DNA-binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb+ control, and both the ssbW54S- and ssbH55K-containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb-1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.

Amino Acid Sequence↗

PubMed1991

Amino acid 55 plays a central role in tetramerization and function of Escherichia coli single-stranded DNA binding protein.

The histidine at position 55 of the amino acid sequence of the Escherichia coli single-stranded DNA binding protein was replaced by tyrosine, glutamic acid, lysine, phenylalanine, and isoleucine. The properties of the mutant proteins were determined using analytical ultracentrifugation, NMR spectroscopy, gel filtration, and fluorimetric detection of their single-stranded DNA binding ability. While the phenylalanine and isoleucine substitutions did not change the properties of the protein measurably, tyrosine and lysine mutants dissociate into subunits and loose some of their binding affinity for poly(dT). For the lysine mutant we show by electron microscopy that the protein, although fully dissociated and possibly denatured in the free state, binds to poly(dT) as a tetramer indistinguishable from the wild-type protein. The process of tetramerization as observed via single-stranded DNA binding ability is composed of a variety of steps ranging in time from some milliseconds to several hours; it probably involves several forms of dissociated and non-native protein.

DNA, Single-Stranded↗