title: A new protein domain for binding to DNA through the minor groove.
abstract: Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Freire
authors.forename: M
authors.initials: M
authors.lastname: Salas
authors.forename: J M
authors.initials: JM
authors.lastname: Hermoso
mesh_terms: D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000254:growth & development*; D001412:Bacillus subtilis; D001667:Binding, Competitive; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004214:Distamycins / Q000494:pharmacology; D005976:Glutaral; D007364:Intercalating Agents / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004214:Distamycins; D007364:Intercalating Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014764:Viral Proteins; C080198:viral protein p6; C030000:stallimycin; D005976:Glutaral