Biochemical characterization of the structure-specific DNA-binding protein Cmb1 from Schizosaccharomyces pombe.
Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation. It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution. Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested. As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor. Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch. Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding. Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region. The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA. The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling. The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA. Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent. In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA.
Sassoon
J
J
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, Berne, 3012, Switzerland. sassoon@ibc.unibe.ch
Lilie
H
H
Baumann
U
U
Kohli
J
J
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch / Q000235:genetics; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005976:Glutaral / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000166:cytology / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013816:Thermodynamics; D014461:Ultracentrifugation
C115714:Cmb1 protein, S pombe; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006609:High Mobility Group Proteins; D008024:Ligands; D029702:Schizosaccharomyces pombe Proteins; D012996:Solutions; D004247:DNA; D005976:Glutaral