Saccharomyces cerevisiae DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol δ and Pol ε in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol ε has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol δ has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol δ and Pol ε was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol δ and Pol ε on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol ε when compared to Pol δ. We conclude that Pol ε and Pol δ exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.

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Four collections of Gal alpha1-4Gal derivatives were synthesised and evaluated as inhibitors of the PapG class II adhesin of uropathogenic Escherichia coli and of the P(N) and P(O) adhesins of Streptococcus suis strains. Galabiosides carrying aromatic structures at C1, methoxyphenyl O-galabiosides in particular, were identified as potent inhibitors of the PapG adhesin. Phenylurea derivatisation at C3' and methoxymethylation at O2' of galabiose provided inhibitors of the S. suis strains type P(N) adhesin with remarkably high affinities (30 and 50 nM, respectively). In addition, quantitative structure-activity relationship models for E. coli PapG adhesin and S. suis adhesin type P(O) were developed using multivariate data analysis. The inhibitory lead structures constitute an advancement towards high-affinity inhibitors as potential anti-adhesion therapeutic agents targeting bacterial infections.

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The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K(d), of 0.12 microM, which is 700 and 10-20 times lower than the K(d) values for the synthetic peptide and the fusion proteins, respectively. However, when an alpha-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K(d) decreased to 0.64 microM. The differences in the K(d) values were mainly governed by differences in the association rate constants (k(ass)). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s(-)(1)), and a very high association rate constant (8 x 10(5) M(-)(1) s(-)(1)), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an alpha-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.

An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.

Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a welldefined three-dimensional structure, which probably contains a-helical elements and is responsible for the tetramerization of the protein.In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic a-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.Keywords: carbonic anhydrase 11; extracellular superoxide dismutase; heterologous expression; site-directed mutagenesis; subunit interaction; tetramer contact area Mammalian cells produce two different types of CuZnSODs, an intracellular and an extracellular form, which are almost equally efficient as catalysts and have nearly identical kinetic parameters (Tibell et al., 1987). The two enzyme forms exhibit significant sequence similarity, but they differ in several respects. The intracellular human CuZnSOD contains 153 amino acid residues per subunit, has a subunit molecular mass of 16 kDa, and is a dimer. The mature human EC-SOD subunit, on the other hand, contains 222 amino acid residues (plus an 18-residue-long signal peptide) and a N-linked oligosaccharide, resulting in a calculated molecular mass of 26.7 kDa (24.2 kDa from the polypeptide and 2.5 kDa from the oligosaccharide) (Edlund et al., 1992). The hEC-SOD protein forms a tetramer, is secreted, and binds to proteoglycans, which are found on the surface of almost every cell in the human body. The central part of the hEC-SOD sequence is homologous with the sequence of the intracellular CuZnSODs, including those amino acid residues that are metal ligands. The 48 N-terminal Reprint requests to: LenaA.E. Tibell, Department of Biochemistry, Umei University, S-901 87 Umei, Sweden; e-mail: lenat@chem.umu.se.Abbreviarions: CuZnSOD, Cu-and Zn-containing intracellular superoxide dismutase; EC-SOD, extracellular superoxide dismutase; GuHC1, guanidine hydrochloride; HCAII, human carbonic anhydrase 11: MRW, mean residue weight: SOD, superoxide dismutase. amino acid residues of hEC-SOD, and 30 of the C-terminal, have no counterparts in intracellular CuZnSODs, and no significant homology with any other sequenced protein.The three-dimensional structure of the intrace...