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A bifunctional DNA binding region in Tn5 transposase.

Gradman RJ, Ptacin JL, Bhasin A, Reznikoff WS, Goryshin IY - Mol. Microbiol. (2007)

Bottom Line: In particular, there is little evidence for the positioning of donor DNA and target DNA.In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex.Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase-DNA binding events.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
Tn5 transposition is a complicated process that requires the formation of a highly ordered protein-DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein-DNA contacts, although many protein-DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA. In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex. Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase-DNA binding events.

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Related in: MedlinePlus

In vivo/in vitro target insertion profile library. The DNA insertion profile library (as described in Fig. 2C) was analysed via 6% denaturing PAGE. Each insertion profile library generated a pattern of bands representing individual insertion events. The specific insertion into the designed 9 bp insertion site is shown. The insertion profile window is ∼300 bp in length. The peak height of the specific insertion event was normalized for each mutant Tnp to the peak height of the control specific event which corresponds with peak 53. Note the large insertion density present at peak 20. This peak corresponds with an insertion site native to plasmid pGRT2 that was not designed. This native site shares sequence elements with the designed 9 bp target site (GGTTATAGG). The location of this native insertion site is denoted in Fig. 2A. A decrease in the secondary peaks as compared with the specific insertion peak represents an increase in target specificity. K164A, R189C and R210A led to large increases in specificity. K212M led to a slight, but reproducible decrease in specificity and an increase in random insertion events (over the span of this insertion window).
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fig03: In vivo/in vitro target insertion profile library. The DNA insertion profile library (as described in Fig. 2C) was analysed via 6% denaturing PAGE. Each insertion profile library generated a pattern of bands representing individual insertion events. The specific insertion into the designed 9 bp insertion site is shown. The insertion profile window is ∼300 bp in length. The peak height of the specific insertion event was normalized for each mutant Tnp to the peak height of the control specific event which corresponds with peak 53. Note the large insertion density present at peak 20. This peak corresponds with an insertion site native to plasmid pGRT2 that was not designed. This native site shares sequence elements with the designed 9 bp target site (GGTTATAGG). The location of this native insertion site is denoted in Fig. 2A. A decrease in the secondary peaks as compared with the specific insertion peak represents an increase in target specificity. K164A, R189C and R210A led to large increases in specificity. K212M led to a slight, but reproducible decrease in specificity and an increase in random insertion events (over the span of this insertion window).

Mentions: A. This assay utilizes the rescue of a TetR gene to measure transposition frequency and the rescue of a LacZ gene (that is missing a start codon) to measure specific insertion events into a designed, preferable 9 bp target site (GCTCAGAGC). Insertions into this 9 bp target site corresponds to peak 53 in Fig. 3. A second preferable target site is native to plasmid pGRT2 and is noted as peak 20 (see Fig. 3). These experiments were repeated twice at multiple dilutions for each mutant Tnp.


A bifunctional DNA binding region in Tn5 transposase.

Gradman RJ, Ptacin JL, Bhasin A, Reznikoff WS, Goryshin IY - Mol. Microbiol. (2007)

In vivo/in vitro target insertion profile library. The DNA insertion profile library (as described in Fig. 2C) was analysed via 6% denaturing PAGE. Each insertion profile library generated a pattern of bands representing individual insertion events. The specific insertion into the designed 9 bp insertion site is shown. The insertion profile window is ∼300 bp in length. The peak height of the specific insertion event was normalized for each mutant Tnp to the peak height of the control specific event which corresponds with peak 53. Note the large insertion density present at peak 20. This peak corresponds with an insertion site native to plasmid pGRT2 that was not designed. This native site shares sequence elements with the designed 9 bp target site (GGTTATAGG). The location of this native insertion site is denoted in Fig. 2A. A decrease in the secondary peaks as compared with the specific insertion peak represents an increase in target specificity. K164A, R189C and R210A led to large increases in specificity. K212M led to a slight, but reproducible decrease in specificity and an increase in random insertion events (over the span of this insertion window).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229646&req=5

fig03: In vivo/in vitro target insertion profile library. The DNA insertion profile library (as described in Fig. 2C) was analysed via 6% denaturing PAGE. Each insertion profile library generated a pattern of bands representing individual insertion events. The specific insertion into the designed 9 bp insertion site is shown. The insertion profile window is ∼300 bp in length. The peak height of the specific insertion event was normalized for each mutant Tnp to the peak height of the control specific event which corresponds with peak 53. Note the large insertion density present at peak 20. This peak corresponds with an insertion site native to plasmid pGRT2 that was not designed. This native site shares sequence elements with the designed 9 bp target site (GGTTATAGG). The location of this native insertion site is denoted in Fig. 2A. A decrease in the secondary peaks as compared with the specific insertion peak represents an increase in target specificity. K164A, R189C and R210A led to large increases in specificity. K212M led to a slight, but reproducible decrease in specificity and an increase in random insertion events (over the span of this insertion window).
Mentions: A. This assay utilizes the rescue of a TetR gene to measure transposition frequency and the rescue of a LacZ gene (that is missing a start codon) to measure specific insertion events into a designed, preferable 9 bp target site (GCTCAGAGC). Insertions into this 9 bp target site corresponds to peak 53 in Fig. 3. A second preferable target site is native to plasmid pGRT2 and is noted as peak 20 (see Fig. 3). These experiments were repeated twice at multiple dilutions for each mutant Tnp.

Bottom Line: In particular, there is little evidence for the positioning of donor DNA and target DNA.In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex.Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase-DNA binding events.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
Tn5 transposition is a complicated process that requires the formation of a highly ordered protein-DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein-DNA contacts, although many protein-DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA. In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex. Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase-DNA binding events.

Show MeSH
Related in: MedlinePlus