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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 pro-foundly 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 | iFEBS Journal Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA Mariana F. Tioni Ivana L. Viola Raquel L. Chan and Daniel H. Gonzalez Catedra de Biologia Celulary Molecular Facultad de Bioquimica y Ciencias Biologicas Universidad Nacional del Litoral Santa Fe Argentina Keywords DNA-binding specificity footprinting homeodomain KNOX protein recognition code Correspondence D. H. Gonzalez Catedra de Biologia Celular y Molecular Facultad de Bioquimica y Ciencias Biologicas UNL CC 242 Paraje El Pozo 3000 Santa Fe Argentina Fax Tel 54 342 4575219 E-mail dhgonza@fbcb.unl.edu.ar Received 13 July 2004 revised 31 August 2004 accepted 21 September 2004 doi 10.1111 j.1432-1033.2004.04402.x 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 lysine shifted the preference to TGACC suggesting that residue 50 interacts with nucleotide s 3 to GAC. Mutation of Lys54 fi Val produced a protein with reduced affinity and relaxed specificity able to recognize the sequence TGAAA while the conservative change of Arg55 fi Lys completely abolished binding to DNA. Based on these results we propose a .
