A new protein domain for binding to DNA through the minor groove

Abstract

Protein p6 of the Bacillus subtilis phage ∅29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of ∅29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic α-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative α-helix. DNA binding and activation of initiation of ∅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 ~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 α-helical content when bound to DNA. All these results indicate that an α-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.