Biomedical subjects

Laura Manelyte

Publications and source records attributed to Laura Manelyte.

4 recordsLinked to original sources

PubMed2006

Structural and functional analysis of the MutS C-terminal tetramerization domain.

The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801-853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an alpha-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutS(CF/D835R) is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1-800), which lacks the CTD. Moreover, the dimer-forming MutS(CF/D835R) has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.

Amino Acid Sequence↗

PubMed2006

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.

To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.

Adenosine Triphosphatases↗

PubMed2004

Mapping protein-protein interactions between MutL and MutH by cross-linking.

Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.

Adenosine Triphosphatases↗

PubMed2003

Solubility engineering of the HhaI methyltransferase.

DNA methylation is involved in epigenetic control of numerous cellular processes in eukaryotes, however, many mechanistic aspects of this phenomenon are not yet understood. A bacterial prototype cytosine-C5 methyltransferase, M.HhaI, serves as a paradigm system for structural and mechanistic studies of biological DNA methylation, but further analysis of the 37 kDa protein is hampered by its insufficient solubility (0.15 mM). To overcome this problem, three hydrophobic patches on the surface of M.HhaI that are not involved in substrate interactions were subjected to site-specific mutagenesis. Residues M51 or V213 were substituted by polar amino acids of a similar size, and/or the C-terminal tetrapeptide FKPY was replaced by a single glycine residue (Delta324G). Two out of six mutants, delta324G and V213S/delta324G, showed improved solubility in initial analyses and were purified to homogeneity using a newly developed procedure. Biochemical studies of the engineered methyltransferases showed that the deletion mutant delta324G retained identical DNA binding, base flipping and catalytic properties as the wild-type enzyme. In contrast, the engineered enzyme showed (i) a significantly increased solubility (>0.35 mM), (ii) high-quality 2D-[(15)N,(1)H] TROSY NMR spectra, and (iii) (15)N spin relaxation times evidencing the presence of a monomeric well-folded protein in solution.

Amino Acid Substitution↗