p element transposition in vitro proceeds by a cut-and- paste mechanism and uses gtp as a cofactor...

P Element Transposition In P Element Transposition In Vitro Proceeds by a Cut-and-Vitro Proceeds by a Cut-and-

Paste Mechanism and Uses GTP Paste Mechanism and Uses GTP as a Cofactoras a Cofactor

David CoatesDavid Coates

IntroductionIntroduction


• Elucidate the mechanism of P Element transposition in vitro



• Examine the effects of manipulating transposition variables (donor, tposase, etc.)




• Find optimal mechanism




• Find optimal mechanism

• Possible result of being able to insert sequences in vitro

Genetic Assay for P Element Genetic Assay for P Element Transposition In VitroTransposition In Vitro

• Overview: Recombinant P Element constructed with tetr gene between P Element Terminal sequences, designated P1-Pwt-tet.

• Tetrameric target used because of its simple restriction enzyme clevage pattern. The target carried the ColE1 plasmid replication origin and the ampr ampicillin resistance gene.

• The donor was used because of its complete lack of homology with the target plasmid.

Genetic Assay for P Element Genetic Assay for P Element Transposition In VitroTransposition In Vitro

• Transposase was bound, then target DNA added, and the results of the reaction were treated with proteinase K and DNA extracted with phenol. The purified DNA was then electroporated into E. coli and plated on ampicillin agar and ampicillin/tetracycline agar. Ratio of amprtetr colonies /ampr colonies taken.

Biochemical Requirements for Biochemical Requirements for Transposition In VitroTransposition In Vitro

• It was unclear whether other proteins were required for transposition. The T0.3 fraction (the protein fraction highly enriched for P Element transposase) was tested in the presence and absence of Drosophila tissue culture cell nuclear and cytoplasmic extracts.

• Inclusion of these extracts did not significantly stimulate the amount of transposition above that of T0.3 alone.

• These extracts were omitted from any further experiments.

• All four rNTPs and dNTPs added in the presence of Mg2+ increased the number of transposition events dramatically. Stepwise omission of different components showed that only GTP and Mg2+ were require for this level of increased activity.


Reaction Donor Transposase amprtetr/ampr x 105 NrNTPs + dNTPs WT + 19 ± 6.4 3rNTPs WT + 20 ± 4.5 3No Nucleotides WT + 1.0 ± 1.0 6

2mM GTP - Mg2+ WT + 0.5 ± 0.2 32mM GTP WT + 15 ± 4.3 112mM GTP WT - <0.3 ± 0.3 72mM GTP Mutant #26 + 0.5 ± 0.4 32mM GTP Mutant #26 - 0.6 ± 0.4 3


• Efficient transposition in vitro required transposase and wild-type DNA sequences at the 3’ end of the P Element. Transposase itself causes a 50 fold increase in transposition within wild-type DNA sequences and the wild-type sequence accounts for a 30 fold increase over Mutant #26.


Reaction Donor Transposase amprtetr/ampr x 105 NrNTPs + dNTPs WT + 19 ± 6.4 3rNTPs WT + 20 ± 4.5 3No Nucleotides WT + 1.0 ± 1.0 6

2mM GTP - Mg2+ WT + 0.5 ± 0.2 32mM GTP WT + 15 ± 4.3 112mM GTP WT - <0.3 ± 0.3 72mM GTP Mutant #26 + 0.5 ± 0.4 32mM GTP Mutant #26 - 0.6 ± 0.4 3

Structure of the Transposition Structure of the Transposition ProductsProducts

• Normally, P Element transposition is accompanied by the generation of 8bp target site duplications flanking the transposon insertion. Restriction enzyme digestion and DNA sequencing indicate which products follow this rule.

• One out the 17 products obtained in the presence of transposase, wild-type donor DNA, and nucleotides had the flanking sequence of the unreacted donor DNA. This product retained the chloramphenicol-resistance gene. The frequency of this happening (1/17 or 0.6) was approximately equal to the background (2mM GTP, wild-type donor DNA, and no transposase) from the first experiment (0.3 ± 0.3).


• When either magnesium or transposase was omitted from the reaction, some aberrant products were obtained that had 12 bp IRs flanking the P element. Other abnormal products were observed in the absence of transposase and if Mutant #26 donor DNA was used.

• Another method used was BamHI digestion. Authentic transposition products give a 2.9kb and a 1.0 kb fragment when digested by BamHI. This only occurred when transposase, magnesium, and GTP were used.

Analysis of Reaction ConditionsAnalysis of Reaction Conditions• Reaction conditions were varied to optimize transposition, including

time, temperature, and GTP concentration.

• Temperatures from 15ºC to 30ºC resulted in normal products, with 30ºC the optimal temp. Above 37º many products had aberrant products.

• Optimal Conditions are as long as possible at 30ºC with excess GTP.

Nucleotide RequirementsNucleotide Requirements

• Various nucleotide effects on transposition were measured.

• At 5µM, GTP was the only natural nucleotide to raise transposition levels above the background.

• The nonhydrolyzable analogs, GMP-PNP and GMP-PCP gave similar levels suggesting that transposition is not obligately coupled to hydrolysis of the phosphate bond.

GTP

GMP-PNP (GMP-PCP similar)


• At 2mM both GDP and UTP yielded about 1/3 the level of 2mM GTP. This may be due however to minor GTP contaminants or weaker interaction of these nucleotides with the transposition machinery.

• The portion of transposition in vitro did not require extensive DNA synthesis as addition of dideoxynucleotides did not inhibit the process.


Nucleotide amprtetr/ampr x 105 N5µM GTP 5.7 ± 1.1 45µM GTP, mutant 26 0.5 ± 0.4 35µM ATP 1.0 ± 0.3 35µM CTP 0.6 ± 0.3 35µM UTP <0.6 ± 0.2 35µM GMP-PNP 5.5 ± 1.6 35µM GMP-PCP 3.9 ± 0.6 35µM GDP 0.3 ± 0.1 35µM dGTP 4.3 ± 1.4 35µM ddGTP 3.5 ± 0.4 32mM GMP <0.8 ± 0.2 32mM GDP 1.0 ± 0.5 3100µM all four dNTPs 9.1, 11 2100µM GTP + 100 µM all 4 ddNTPs 24, 15 2

Topological State of Donor DNATopological State of Donor DNA

• The ability of P1-Pwt-tet to function when linearized was examined.

• Restriction sites (PstI) far from the P Element were cleaved and the DNA was linearized.

• This linearization did not markedly affect the frequency of the transposition. BamHI analysis and sequencing indicated correct products. This indicates donor supercoiling is negligible.

• Target plasmids treated with DNAase I during nick translation were used in in vitro reaction with roughly the same efficiency, indicating target supercoiling is not necessary either.

Donor Plasmid Cleavage Site Postcleavage Treatment amprtetr/ampr x 105 NP1-pwt-tet none 15 ± 4.3 11P1-pwt-tet PstI 9.5, 11 2

Structural Requirements at the P Structural Requirements at the P Element DNA TerminiElement DNA Termini

• XbaI cleavage of the XB+4 constructs yielded P Element DNA fragments that had at least 12 nucleotides of flanking DNA on both strands of both termini. These were comparable in activity to normally cleaved donors (350 bp lead ins)

• BsaI cleavage yielded fragments with four flanking nucleotides on the 5’ terminus and an exposed 3’ OH. These were inefficient donors.


• SacI plasmid cleavage yielded P element DNA fragments that had four flanking nucleotides at the 3’ terminus and an exposed 5’ phosphate on the terminal P element nucleotide. These were very effective donors.

• Removal of the 4 nucleotide 3’ extension by treatment with T4 DNA polymerase reduced the reactivity.


Donor Plasmid Cleavage site Postcleavage treatment amprtetr/ampr x 105 NP1-Pwt-tet none 15 ± 4.3 11P1-Pwt-tet PstI 9.5, 11 2XB + 4 wt XbaI 14, 12 2XB + 4 wt BsaI 0.6 ± 0.3 3XB + 4 26 XbaI <0.3 1XB + 4 26 BsaI <0.2, <0.2 2SacI wt SacI 46 ± 25 10SacI wt SacI T4 Dna polymerase 1.7 ± 0.4 6SacI wt SacI CIP 77 ± 15 6SacI wt SacI CIP, PNK 58 ± 13 6SacI 26 SacI 1.3 ± 0.7 7SacI 26 SacI T4 Dna polymerase <0.3, <0.3 2SacI 26 SacI CIP 0.9, 1.1 2SacI 26 SacI CIP, PNK 1.4, 3.5 2XB wt BsaI <0.2 ± 0.1 5XB wt BsaI Klenow + 4 dNTPs 2.4 ± 1.3 10XB wt BsaI Klenow + 3dNTPs ddGTP <0.2 ± 0.1 4XB wt BsaI Klenow + dATP, dCTP, TTP <0.3, <0.3 2XB 26 BsaI <0.2 ± 0 4XB 26 BsaI Klenow + 4 dNTPs <0.5 ± 0.2 7XB 26 BsaI Klenow + 3dNTPs ddGTP <0.3 ±0.2 4XB 26 BsaI Klenow + dATP, dCTP, TTP <0.3, 0.4 2PCR wt Klenow + 4 dNTPs 1.5 ± 0.3 4PCR 26 Klenow + 4 dNTPs <0.4 ± 0.1 4


• Suspecting an increase in activity in the presence of the 5’-terminal phosphate groups in transposition, SacI cleaved donors were treated with CIP (calf intestinal phosphotase) to remove the phosphate group. The absence or presence of the 5’-terminal phosphate group did not substantially change the reactivity of the donor DNA.

• PCR P Elements were also created to test the reactivity of P Elements without the 5’-terminal phosphate. With a lead in of 20 nucleotides to the P Element, the PCR products were approximately as effective as the SacI products treated with T4 DNA polymerase (blunt ends).


• Cleavage of the XB plasmids with BsaI gave P Elements with an exposed 5’-terminal P Element nucleotide and a four-nucleotide recessed 3’ terminus. With no additional treatment, neither the wild or mutant donor yielded transposition products.

• Blunt-ending with Klenow polymerase increased the product in wild donors to T4 DNA Polymerase-treated levels. Mutant donors never functioned.

DiscussionDiscussion

• Transposition depends on the addition of partially purified P Element Transposase



• P Element transposition in vitro generated the classic in vivo 8bp target site duplication.



• P Element transposition in vitro generated the classic in vivo 8bp target site duplication.

• Transposition was greatly diminished by the deletion of the transposase binding site at the 3’ terminus.


• It is possible that a covalent protein-DNA intermediate is involved in transposition, but such a structure could not conserve the bond energy of the broken donor DNA strands.

DiscussionDiscussion• Though some transposition mechanisms

include formation of a cointegrate intermediate in which donor and target DNA is flanked by 2 transposon copies in a direct repeat fashion, the data complements genetic evidence suggesting that P Elements transpose by a cut-and-paste mechanism, leaving a double-stranded gap that is then repaired using the homologous chromosome as template.

• The cut-and-paste mechanism is particularly supported by the linearization experiment. The cointegrate model cannot occur if both strands are cleaved before transposition.


• Ultra purified transposase preparations do not produce transposition, indicating that there is some manner of accessory protein, but all attempts at determining the stimulatory protein have been unsuccessful.

• Most DNA rearrangement reaction require ATP. P Element transposition is unique in that it uses GTP. It is still unknown how it affects transposition though, whether it interacts with the transposase itself or it affects the formation of higher order nucleoprotein complexes by transposase. It seems apparent that GTP hydrolysis is not necessary though. This may be an oversight of the in vitro system, with NTP hydrolysis important for steps leading to strand transfer. GTP may simply be a signal that couples metabolism to the stimulation of P Element transposition.


• Ideal conditions appear to be

Transposase inducedWild Type Donor30ºC tempSacI digest treated with CIPRun as long as possibleWith excess GTP


• Problems with the paper:Mainly the number of trials for each experiment. Most were three, many twos and a one.

• Uses:The specific and rapid incorporation of P Elements into targets, and ways to increase product and precision in an in vitro system.

p element transposition in vitro proceeds by a cut-and- paste mechanism and uses gtp as a cofactor...

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