Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA

TIONI, MARIANA F.; VIOLA, IVANA L.; CHAN, RAQUEL L.; GONZALEZ, DANIEL H.

FEBS JOURNAL

JOHN WILEY & SONS INC

Año: 2005 vol. 272 p. 190 - 202

1742-464X

The interaction of the homeodomain of the sunflower KNOX protein HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl radical footprinting and missing nucleoside experiments. Binding of HAKN1 to different oligonucleotides indicated that HAKN1 prefers the sequence TGACA (TGTCA), with changes within the GAC core more profoundly affecting the interaction. Footprinting and missing nucleoside experiments using hydroxyl radical cleavage of DNA showed that HAKN1 interacts with a 6-bp region of the strand carrying the GAC core, covering the core and nucleotides towards the 3’ end. On the other strand, protection was observed along an 8-bp region, comprising two additional nucleotides complementary to those preceding the core. Changes in the residue present at position 50 produced proteins with different specificities. An I50S mutant showed a preference for TGACT, while the presence of Lys shifted the preference to TGACC, suggesting that residue 50 interacts with nucleotide(s) 3’ to GAC. Mutation of Lys54 to Val produced a protein with reduced affinity and relaxed specificity, able to recognise the sequence TGAAA, while the conservative change of Arg55 to Lys completely abolished binding to DNA. Based on these results, we propose a model for the interaction of HAKN1 with DNA in which helix III of the homeodomain accommodates along the major groove with Arg55, Asn51, Lys54 and Ile50, respectively, establishing specific contacts with bases of the GACA sequence or its complements. This model can be extended to other KNOX proteins given the conservation of these amino acids in all members of the family.