Determination of the DNA binding properties of HAT3.1, a plant protein with a non-canonical homeodomain.

IVANA L. VIOLA; DANIEL H. GONZALEZ

Pinamar. Buenos Aires, Argentina

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2005

SAIB

HAT3.1 is an Arabidopsis homeodomain protein characterized by an N-terminal domain known as the PHD-finger. The similarity between this motif and the RING-finger domain raises the possibility that HAT3.1 may be involved in either transcriptional activation, recombination or DNA repair. The HAT3.1 homeodomain (HD) differs substantially from those in other HD proteins identified so far, even in positions which are almost invariant or highly conserved. In the present work, we demonstrate that the HAT3.1 HD is able to specifically bind DNA and identify its target sequence by the random binding site selection assay (SELEX). The consensus sequence identified after twelve rounds of binding and amplification differs markedly from those recognized by other HDs. The unusual DNA binding characterisitics of HAT3.1 may be explained by the particular amino acid composition of the HD. Accordingly, several site-directed mutants (H51N, H51A, K50Q and K50A) were constructed to study their DNA binding affinity and specificitty compared with the wild-type protein. In addition, footprinting and missing nucleoside experiments using hidroxyl radical cleavage of DNA were used to obtain a more detailed picture of the binding of the HAT3.1 HD to its target site.