Single Base-pair Precision and Structural Rigidity in a Small IHF-induced DNA Loop

Abstract

The prokaryotic integration host factor (IHF) is a DNA-binding protein that binds to specific DNA sites as a heterodimer. Genetic and mutational analyses have previously identified asymmetric protein-DNA contacts by the individual subunits. By exploiting the unique sequence and positional context of one IHF binding site, H′ in Lambda attachment sites ( attsites), we have identified a symmetry element of binding and have localized the functional bend center to the center of this symmetry. A shift of the H′ bend center by a single base-pair to the right or to the left within the very tight loop formed with Lambda integrase (int) and IHF in att-site “intrasomes” severely reduces recombination. This suggests that a precise, but wrongly positioned, DNA bend within a loop constant length negatively influences the juxtaposition or “phasing” of the core-type and arm-type Int binding sites by differentially affecting the length of each leg of the loop. Furthermore, ten base-pair insertions within this loop that should not interfere with correct helical phasing are sensed in a position-dependent manner. Distal insertions abolish recombination, whereas proximal or double insertions (in both legs of the loop) are well tolerated.