Mutational Analysis of the Base Flipping Event Found in Tn5 Transposition

Abstract

To study the biochemical significance of this phenomenon, we mutated the Tnp residues proposed to be involved in stabilizing this interaction and removed the T2 nucleotide to examine which steps in the transposition reaction require T2-Tnp interactions. From this work, we have determined that stacking interactions between T2 and Tnp are critical for efficient transposition in vitro. In addition, our results suggested that T2-Tnp interactions facilitate hairpin formation and hairpin resolution primarily through base stacking and that T2 plays a role in the alignment of the transposon DNA for strand transfer.Mobile DNA elements share many similar characteristics. The proteins responsible for movement of these elements manifest common mechanistic features and contain considerable structural similarities in their catalytic domains. In the Tn5 system, Tn5 transposase (Tnp) 1 functions as a homodimer and catalyzes all of the steps involved in the movement of Tn5 from one location to another within a genome (3-5).The model for Tn5 transposition is illustrated in Fig. 1. First, it is believed that each Tnp monomer binds to two separate recognition sequences, forming the initial contacts. The monomers then dimerize to form the synaptic complex (6). This complex brings the end sequences proximal to one another. In the synaptic complex, each Tnp monomer binds to both end sequences. The first set of contacts (cis contacts) are maintained predominately between the N-terminal domain and the middle of one end sequence, whereas the second set of Tnp-DNA contacts (trans contacts) are formed primarily between the catalytic domain and the other end sequence (1). This complex is a prerequisite to cleavage, since residues near the trans Tnp-DNA contacts are responsible for strand cleavage (1, 7). The cleavage reaction begins as Tnp nicks the transferred DNA strand at the donor DNA-transposon junction, adjacent to position 1 of the end sequence, leaving a 3Ј OH group. This nick occurs through the nucleophilic attack by an activated water molecule coordinated by Mg 2ϩ . The 3Ј OH group on the transferred strand in turn acts as a nucleophile to cleave the complementary nontransferred DNA strand, forming a hairpin at the transposon end and releasing the donor DNA. Hydrolysis of the hairpin by another activated water molecule, coordinated in the same Tnp monomer, resolves the hairpin and results in the formation of transposon DNA competent for integration (8). The cleaved transposon DNA remains in a nucleoprotein complex called the post-cleavage synaptic complex. This complex binds to the target DNA, forming the target capture complex (9). The transposon DNA contains free 3Ј OH moieties on the transferred strands that attack the phosphodiester backbone of the target DNA in a staggered transesterification reaction termed strand transfer. A 9-bp stagger for the strand transfer step creates two complementary 9-base single-stranded DNA regions flanking the Tnp insertion. These single-stranded DNA regions are repaired by the cell DNA replicat...

Requirements for DNA hairpin formation by RAG1/2

“…In Tn5, the hairpin forms on the transposon end, and in the crystal, a flipped-out base is seen stabilized by a tryptophan (W298). Mutagenesis studies show that this interaction is important for efficient hairpin formation (20). Similar findings were made with mutagenesis of equivalent residues in Tn10 (W265) (21) and Hermes (W319) transposases (22).…”

“…It has been shown in the Tn5 system that rescue of hairpin formation by a Trp mutant involved in base flipping can be achieved by using the appropriate abasic DNA substrate, because there is no base needing to be stabilized (20). We therefore tested for the rescue of transesterification by W760A, W893A, and W956A on both types of flanks in Mn 2ϩ .…”

Diverse DNA transposons in rotifers of the class Bdelloidea

“…However, the use of this TIR sequence, together with the TTAA target, as a PCR primer yielded only a weak and diffuse band, and sequencing of cloned amplicons revealed two defective (one frameshift and two stop codons in each) copies of a related (60% amino acid identity) family, designated AvPB2, which are 97% identical and share an intron in the TPase ORF. A conserved Cys 5 -His-Cys 2 motif at the C terminus of the TPase and the preceding bipartite NLS (19,37) are detectable in AvPB1 and AvPB2, as is the putative DDD catalytic triad, which can be described as DE (77)DN(97)D (2)D. Moreover, a highly conserved tryptophan with adjacent basic residues, a motif essential for base flipping during hairpin formation and resolution in Tn5 and possibly in hAT and in RAG recombinase (35,40), is found between the catalytic domain and the Cys-rich motif (Fig. 1B), indicating that a hairpin intermediate may be formed during piggyBac transposition.…”

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

“…Structural analysis of the Tn5 transposase (27,28) coupled with biochemical and genetic experiments on both Tn5 (29,30) and Tn10 (31) has defined a hairpin binding module in these transposases (32). Because telomere resolution by ResT also involves the formation of a DNA hairpin, it seemed reasonable to examine the ResT sequence for the hairpin binding region found in the Tn5 and Tn10 transposases.…”

“…1A) (27,28,32). Some of the mutations in this region result in a transposase that cannot form the hairpin intermediate and therefore results in the accumulation of nicks at the transposon ends (29)(30)(31). Between the hydrophobic binding pocket and the YREK motifs is an intervening stretch of 18 or 19 intervening amino acids that are not part of the hairpin binding domain (Fig.…”