1. Excision of the Drosophila Mariner Transposon Mos1
Angela Dawson, David J Finnegan 
Molecular Cell 
Volume 11, Issue 1, Pages (January 2003)
DOI: /S (02)

2. Figure 1
Mos1 Transposase Cleaves to Generate Three Base Staggered Breaks
(A) Primer extension analysis of Mos1 excision in vitro. Linear PCR extension was performed on the reaction products generated following excision in the presence of 2.5 mM Mn2+ using pMos plasmid as a donor. Mn2+ was used in this reaction to enhance cleavage at IRL and may have increased cleavage at secondary sites. Sequence reactions (GATC) were loaded as markers. PCR was with primer 1 (Z7401) to map the site of 5′ cleavage or primer 2 (Z7601) to map the site of 3′ cleavage. The sequence at the site of cleavage is shown below. The cleavage sites are shown with arrows.
(B) Substrate IRR100 used to investigate cleavage in vitro. Strand 1 is the transferred strand and strand 2 the nontransferred strand. Upper case letters indicate the terminal DNA sequence of the right hand of Mos1. Lower case letters indicate flanking DNA. The TA dinucleotide flanking Mos1 is in bold face. Numbers indicate base pair positions relative to the transposon-donor junction.
(C) Analysis of oligonucleotide cleavage products by denaturing gel electrophoresis. The position of the 32P label is marked by an asterisk. IRR100 labeled on either the nontransferred strand (lanes 1 and 2) or on the transferred strand (lanes 4 and 5) was incubated with (lanes 2 and 5) or without (lanes 1 and 4) Mos1 transposase under standard conditions. A 33-mer (the product of cleavage at the +3 position of the nontransferred strand) and a 70-mer (the product of cleavage precisely at the end of the element on the transferred strand) oligonucleotides were included as markers (lanes 3 and 6, respectively).
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2 Kinetics of IRR100 Cleavage by Mos1 Transposase
(A) Cleavage of the nontransferred strand was detected using IRR100 labeled at the 5′ end of the flanking DNA. An asterisk indicates the position of the radioactive label. Reactions were assembled at room temperature under standard conditions, and the reaction was initiated by the addition of transposase (to a final concentration of 7 nM). At the indicated time points, reactions were terminated by addition of EDTA to 20 mM, an equal volume of STOP buffer was added, and a proportion of the reaction was applied to a 6% denaturing polyacrylamide gel.
(B) Cleavage of the transferred strand was detected using IRR100 labeled on the 3′ end of the flanking DNA. Samples were processed as for (A).
(C) Detection of double-strand breaks. Identical results were obtained using substrate labeled on either strand. IRR100 labeled on the 5′ end of the transferred strand is shown here. Reactions were terminated by the addition of EDTA and 0.5% SDS. Samples were applied to a native 6% polyacrylamide gel.
(D) Graphical representation of the data from (A)–(C). Values are expressed as the percentage of the amount of cleavage of the initial substrate.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
The Effect of ddA at the Site of First Strand Cleavage on Second Strand Nicking
Substrates were assembled that contained a break at the site of first strand cleavage (+3 on the nontransferred strand) and with either 3′ dA or 3′ ddA at this position. Reactions were incubated with transposase (lanes 1, 2, 5, and 6) or without transposase (lanes 3, 4, 7, and 8) in the presence of 5 mM MgCl2. Following incubation for 2 hr at 30°C, reactions were terminated by the addition of EDTA to 20 mM. Substrates labeled on the nontransferred strand were separated under native conditions (lanes 1–4). Samples labeled on the transferred strand of the Mos1 sequence were separated under denaturing conditions (lanes 5–8) as described previously.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4 An Aberrant Hairpin Is Detected under Some Conditions
(A) A time course of transposase-mediated second strand cleavage under nonstandard conditions. IRR100 precleaved at the site of first strand cleavage was incubated with transposase for the time indicated. Reactions were initiated by the addition of transposase (to a final concentration of 459 nM), with Mn2+ (2.5 mM) present as divalent cation. Reactions were terminated by the addition of EDTA and STOP buffer. Samples were applied to a 6% denaturing gel, which was run at 60°C. An arrow indicates the position of the hairpin DNA. The original 33-mer radiolabeled oligonucleotide substrate is indicated.
(B) Behavior of the high molecular weight band following elution and separation by native and denaturing electrophoresis. DNA was eluted overnight from the gel (A) and rerun under native (lane 1) or denaturing conditions (lane 3). Radiolabeled MspI-digested pUC18 DNA was included as a marker (lanes 2 and 4).
(C) The high molecular weight species was isolated from a denaturing polyacrylamide gel and subjected to an A+G Maxam-Gilbert sequencing reaction (lane 1). Lane 2 shows the result of performing the same reaction on the original 33-mer oligonucleotide substrate. The sequence of the starting substrate is indicated at the bottom with a line showing where the two strands have been joined to form a hairpin. The arrowhead indicates the position of the nick in the bottom strand of the substrate.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 Mos1 Transposase Forms a Paired End Complex
(A) Demonstration of a PEC. Reactions were assembled under catalytic conditions for 2 hr in the presence of 0.1 mM MgCl2. Reactions containing combinations of IRR100 and/or IRR80 as indicated were incubated with transposase under standard conditions in the presence of Mg2+ and DMSO (lanes 1–4) or in the absence of DMSO (lane 5) or Mg2+ (lane 6). The position of the 32P is marked by an asterisk. Presumptive DNA species contained in the observed complexes are indicated.
(B) Flanking DNA is released during the cleavage reactions. Reactions were carried out using IRR100 labeled on either the transferred strand (lanes 1 and 2) or the nontransferred strand (lanes 4 and 5). Following incubation under standard catalytic conditions, samples were applied directly (lanes 1 and 4) or following deproteinization (lanes 2 and 5) to a native gel. PEC and STC complexes are indicated. Lanes 3 and 6 contain DNA markers identical to the proposed double-strand products of cleavage of IRR100.
(C) A low mobility complex is detectable which contains the products expected of strand transfer into unreacted IRR100. STC complex formed with IRR100 labeled on the transferred strand was eluted overnight in activity buffer with 10 mM EDTA, and protein was removed by phenol extraction. The purified DNA was analyzed on a 6% denaturing gel (lane 1). Lanes 2 and 3 contain DNA markers of 100 nucleotides corresponding to a full-length IRR100 strand and of 70 nucleotides representing the labeled fragment of the transferred strand after cleavage.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Mutation of the CA Dinucleotide Flanking the Site of First Strand Cleavage Inhibits Second Strand Cleavage and PEC Formation
The CA dinucleotide was changed to GG as indicated at the top of the figure. Cleavage reactions were performed using wild-type (lanes 1, 3, 5, and 7) and mutant substrate (lanes 2, 4, 6, and 8) under standard conditions with (lanes 1, 2, 5, and 6) or without transposase (lanes 3, 4, 7, and 8) as indicated. Oligonucleotide cleavage products produced by cleavage of the nontransferred strand are shown in lanes 1–4. Products corresponding to cleavage of the transferred strand are shown in lanes 5–8. The positions of the normal cleavage products are indicated. The ability of the wild-type and mutant substrate to form a PEC is compared in lanes 9 and 10, respectively. Arrows indicate the position of PEC and STC complexes.
Molecular Cell  , DOI: ( /S (02) )