Center. Hix Dna Interactions With The Hin Protein Nmr Studies

The Hin recombinase catalyzes a site-specific DNA inversion between two 26 base pairs (bp) inverted sequences (HixL and HixR) that flank a 993-bp DNA segment, which regulates the alternate expression of the flagellin genes in Salmonella Typhimurium. At the first stage of DNA inversion, the Hin binds to each 26-bp Hix DNA site as a dimer with high affinity (Kd ~ 10-9) and specificity. The origin of this specificity has been understood by the major and minor grooves contacts with the Hin at 9-13 and 5-6 DNA positions, respectively (numbering from the center of the inverted sequence). However, we have found that DNA mutations at the central positions (+1,-1) of the Hix site while other positions having intact sequences are also able to modulate the Hin binding. To investigate an unknown additional specificity factor explaining this result, we have determined the solution structures and dynamics of the wild type and mutant Hix site DNA by NMR spectroscopy and Fluorescence Resonance Energy Transfer (FRET) especially focusing on the central positions. Contributions of the central part of the Hix site to the sequence specific interaction with the Hin in the absence of significant physical contacts will be discussed in a view of deformability of DNA structure.