A new protein domain for binding to DNA through the minor groove.
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.
Freire
R
R
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Salas
M
M
Hermoso
J M
JM
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*
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