Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition

Mei Tang,Ciro Cecconi,Carlos Bustamante,Donald C Rio

doi:10.1074/jbc.m704106200

Abstract

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.

Highlights

Mobile genetic elements are ubiquitous among both prokaryotic and eukaryotic organisms [1]
Whereas some transposon systems have been studied with respect to the assembly state of their transposase, the oligomeric state of the active P element transposase protein and how it initially interacts with P element DNA are unknown
Initial P Element Transposase-DNA Complexes Form at One Transposon End Prior to Synapsis—The P element transposition reaction occurs in stages, with initial transposase binding, synapsis, nonconcerted DNA cleavage, and the target capture and integration (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Protein Standards for Protein Volume Measurements—RNA polymerase holoenzyme was kindly provided by Dr Caroline Kane (University of California, Berkeley). Gel Filtration Chromatography—Approximately 3.5 ␮g of immunoaffinity-purified transposase protein was resolved on a Superdex 200 PC3.2/30 column (GE Healthcare) on a SMART system at 4 °C in HGKED buffer (25 mM Hepes-KOH, pH 7.6, 1 mM EDTA, 300 mM KCl, 0.5 mM DTT, and 10% glycerol) at a flow rate of 40 ␮l/min. Standard proteins used to calibrate the column were bovine serum albumin (66 kDa), catalase (232 kDa), and thyroglobulin (669 kDa) and were run in a parallel gradient in the same buffer as transposase. To compare the apparent sizes of transposase oligomers between gel filtration and glycerol gradient sedimentation fractions, we used the protein peak fractions from both methods and carried out a single end binding experiment where the protein was incubated with DNA substrate DNA in the absence of GTP for 30 min, and the reaction mixture was deposited on mica, processed, and imaged as indicated above and in Ref. 14. Integration of the area of the ellipse along the height axis gave a volume equal to p/6 ϫ (length ϫ width ϫ height)

RESULTS

That Interacts Initially with One

DISCUSSION