Target DNA Bending is an Important Specificity Determinant in Target Site Selection in Tn10 Transposition

Abstract

The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA (∼24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca2+ facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca2+ could, in theory, exert this effect by stabilizing bends in the target DNA.