TN5 TRANSPOSASE MUTANTS THAT ALTER DNA-BINDING SPECIFICITY

Tn5 transposase (Tnp) binds to Tn5 and IS50 end inverted repeats, the outside end (OE) and the inside end (IE), to initiate transposition. W e report the isolation of four Tnp mutants (YH41, TP47, EK54 and EV54) that increase the OF-mediated transposition frequency and enhance the binding affinity of Tnp for OE DNA. In addition, two of the Tnp mutan ts (TP47 and EK54) appear to be change-of-specificity mutants, since t hey alter the recognition of OE versus IE relative to the wild-type Tn p. EK54 enhances OE recognition but decreases IE recognition. TP47 enh ances both OE and IE recognition but with a much greater enhancement f or IE than for OE. This change-of-specificity effect of TP47 is observ ed only when TP47 Tnp is synthesized in cis to the DNA that contains t he ends. We propose that Lys54 makes a favorable interaction with an O F-specific nucleotide pair(s), while Pro47 may cause a more favorable interaction with an IF-specific nucleotide pair(s) than it does with t he corresponding OF-specific nucleotide pair(s). A model to explain th e preference of TP47 Tnp for the IE in cis but not in trans is propose d. (C) 1997 Academic Press Limited.