Crenarchaeal chromatin protein Cren7 binds double-stranded DNA in the minor groove, introducing a sharp single-step DNA kink. The side chain of Leu28, a residue conserved among all Cren7 homologs, intercalates into the kinked DNA step. In the present study, we replaced Leu28 with a residue containing a hydrophobic side chain of different sizes (i.e. L28A, L28V, L28I, L28M and L28F). Both the stability of the Cren7–DNA complex and the ability of Cren7 to constrain DNA supercoils correlated well with the size of the intercalated side chain. Structural analysis shows that L28A induces a kink (∼43°), nearly as sharp as that produced by wild-type Cren7 (∼48°), in the bound DNA fragment despite the lack of side chain intercalation. In another duplex DNA fragment, L28F inserts a large hydrophobic side chain deep into the DNA step, but introduces a smaller kink (∼39°) than that formed by the wild-type protein (∼50°). Mutation of Leu28 into methionine yields two protein conformers differing in loop β3–β4 orientation, DNA-binding surface and DNA geometry in the protein–DNA structure. Our results indicate that side chain intercalation is not directly responsible for DNA kinking or bending by Cren7, but plays a critical role in the stabilization of the Cren7–DNA complex. In addition, the flexibility of loop β3–β4 in Cren7, as revealed in the crystal structure of L28M–DNA, may serve a role in the modulation of chromosomal organization and function in the cell.

You do not currently have access to this content.