Recognition of triple-helical DNA structures by transposon Tn7

Abstract

We have found that the bacterial transposon Tn7 can recognize and preferentially insert adjacent to triple-helical nucleic acid structures. Both synthetic intermolecular triplexes, formed through the pairing of a short triplex-forming oligonucleotide on a plasmid DNA, and naturally occurring mirror repeat sequences known to form intramolecular triplexes or H-form DNA are preferential targets for Tn7 insertion in vitro. This target site selectivity depends upon the recognition of the triplex region by a Tn7-encoded ATP-using protein, TnsC, which controls Tn7 target site selection: the interaction of TnsC with the triplex region results in recruitment and activation of the Tn7 transposase. Recognition of a nucleic acid structural motif provides both new information into the factors that influence Tn7's target site selection and broadens its targeting capabilities.

Figure 1

Cut-and-paste transposition. Recombination initiates by means of double-strand breaks at either end of the transposon (white rectangle) from donor DNA (black lines), exposing terminal 3′-OH groups. Once cut from donor DNA, this excised linear transposon intermediate is joined to the target DNA (gray lines). The 5′ transposon ends are flanked by short gaps; upon repair, a simple insertion product is generated.

Figure 2

Chemical footprinting of the duplex and intermolecular triplex found in pRAO1^dup and pRAO1^tpx, respectively, under in vitro transposition conditions. DMS methylation followed by NaOH treatment of the 5′-end-labeled purine strand generates a guanine and, to a lesser extent, adenine cleavage pattern. (A) The sequence of the triplex region. (B and C) Sequencing gel (B) and line graph (C) analysis of the cleavage pattern reveals the presence of the 15-bp TFO in the major groove; triplex formation results in a corresponding protection from DMS methylation and subsequent cleavage (lane 2) compared with duplex (lane 1). Hypercleavage at the site of psoralen intercalation and crosslinking is seen at the 5′ triplex-duplex junction.

Figure 3

Assay for target specificity. (A) The TnsABC^A225V in vitro transposition reaction generates a population of insertion products in which Tn7 is excised from the donor plasmid (mTn7) and inserted “randomly” into a duplex target plasmid (pRAO1^dup). Target plasmids containing triplex (pRAO1^tpx) generate specific insertion products adjacent to the triplex motif. (B) Unique HindIII sites located inside the transposon and in the target are used to digest reaction products, allowing random and specific insertions to be distinguished. On gel electrophoresis, the population of “random” inserts into pRAO1^dup generates a ladder of products which are visualized by Southern blotting (lane 1). In contrast, an accumulation of specific insertions adjacent to triplex yields two specific products upon digestion (lane 2).

Figure 4

Comparison of insertion profiles of duplex- and triplex-containing targets. (A) Distribution of 100 insertions into pRAO1^dup and pRAO1^tpx. These target plasmids are identical except for a 13-bp TFO annealed and crosslinked to pRAO1^tpx. (B) A second set of targets, pRAO2^dup and pRAO2^tpx, with varied sequence for both plasmid and TFO is shown. For the distribution patterns into the duplex plasmids, each line represents a single insertion; an open circle corresponds to a left-to-right orientation for Tn7, while a closed circle is right-to-left. The origin of replication is drawn in hatched lines. For the triplex-containing plasmids, the nucleotide sequence surrounding the triplex is shown; the number of insertions at each nucleotide is proportional to the length of the lines drawn. More than 70% of all insertions are found within a 40-bp region of each TFO. For these insertions, nearly all (>98%) occurred such that the right end of Tn7 element lands nearest the 5′ end of the triplex. A Tn7 element is drawn above the regional hot-spot to illustrate this orientation bias. Underlined nucleotides within the TFO sequence denote the use of modified bases.

Figure 5

Effect of TFO removal on targeting. (A) Schematic illustrating removal of ribonucleotide-containing TFO upon hydrolysis. C* denotes a ribonucleotide cytosine, which is susceptible to hydrolysis by RNase treatment. Upon hydrolysis, a target substrate with psoralen still crosslinked (p-rRAO1^pso) is generated. These pRAO1^dup, p-rRAO1^tpx, and p-rRAO1^pso plasmids were then brought into the in vitro transposition reaction and assayed for target specificity. (B) A Southern blot using Tn7-specific probes illustrates the loss of targeting adjacent to the triplex upon removal of the TFO. Although the psoralen is still attached, no accumulation of triplex specific inserts was observed in reactions with the p-rRAO1^pso target plasmid (lane 3). The band labeled “Target” is due to background cross-hybridization to linearized target DNA.

Figure 6

Binding activity of transposition proteins: A graph showing relative affinities of TnsA, TnsB, and TnsC^A225V for duplex and triplex DNA. Under in vitro reaction conditions, a titration of TnsC^A225V binding shows a distinct preference for triplex over duplex DNA, whereas TnsA + TnsB shows little binding to either substrate. The percent bound reflects the fraction of the labeled substrate bound by TnsC^A225V.

Figure 7

H-DNA target and Tn7 insertion profile. The 46-bp mirror repeat sequence capable of forming an intramolecular triplex or H-DNA (H-y3 isomer) is illustrated. Distribution of 85 independent insertions isolated by means of transformation were mapped onto pCW2966. An accumulation of insertions is found on either side of the 46-bp mirror repeat sequence; >98% of these insertions occur such that Tn7's right end is closest to the mirror repeat.