Transcript P Element Transposition In Vitro Proceeds by a Cut
P Element Transposition In Vitro Proceeds by a Cut-and-Paste Mechanism and Uses GTP as a Cofactor
David Coates
Introduction
Introduction
• Elucidate the mechanism of P Element transposition in vitro
Introduction
• Elucidate the mechanism of P Element transposition in vitro • Examine the effects of manipulating transposition variables (donor, tposase, etc.)
Introduction
• Elucidate the mechanism of P Element transposition in vitro • Examine the effects of manipulating transposition variables (donor, tposase, etc.) • Find optimal mechanism
Introduction
• Elucidate the mechanism of P Element transposition in vitro • Examine the effects of manipulating transposition variables (donor, tposase, etc.) • Find optimal mechanism • Possible result of being able to insert sequences in vitro
Genetic Assay for P Element Transposition In Vitro
• Overview: Recombinant P Element constructed with tet
r
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 amp
r
ampicillin resistance gene.
• The donor was used because of its complete lack of homology with the target plasmid.
Genetic Assay for P Element Transposition 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 amp
r
tet
r
colonies /amp
r
colonies taken.
Biochemical Requirements for Transposition 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 Mg
2+
increased the number of transposition events dramatically. Stepwise omission of different components showed that only GTP and Mg
2+
were require for this level of increased activity.
Biochemical Requirements for Transposition In Vitro
Reaction rNTPs + dNTPs rNTPs No Nucleotides 2mM GTP - Mg 2+ 2mM GTP 2mM GTP 2mM GTP 2mM GTP Donor WT WT WT WT WT WT Mutant #26 Mutant #26 + + + Transposase amp r tet r /amp r x 10 5 + + + 19 ± 6.4
20 ± 4.5
1.0 ± 1.0
0.5 ± 0.2
15 ± 4.3
<0.3 ± 0.3
0.5 ± 0.4
0.6 ± 0.4
N 3 11 7 3 3 3 3 6
Biochemical Requirements for Transposition In Vitro
• 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.
Biochemical Requirements for Transposition In Vitro
Reaction rNTPs + dNTPs rNTPs No Nucleotides 2mM GTP - Mg 2+ 2mM GTP 2mM GTP 2mM GTP 2mM GTP Donor WT WT WT WT WT WT Mutant #26 Mutant #26 + + + Transposase amp r tet r /amp r x 10 5 + + + 19 ± 6.4
20 ± 4.5
1.0 ± 1.0
0.5 ± 0.2
15 ± 4.3
<0.3 ± 0.3
0.5 ± 0.4
0.6 ± 0.4
N 3 11 7 3 3 3 3 6
Structure of the Transposition Products
• 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).
Structure of the Transposition Products
• 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 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 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 b-g phosphate bond.
GTP GMP-PNP (GMP-PCP similar)
Nucleotide Requirements
• 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 Requirements
Nucleotide 5µM GTP 5µM GTP, mutant 26 5µM ATP 5µM CTP 5µM UTP 5µM GMP-PNP 5µM GMP-PCP 5µM GDP 5µM dGTP 5µM ddGTP 2mM GMP 2mM GDP 100µM all four dNTPs 100µM GTP + 100 µM all 4 ddNTPs amp r tet r /amp r x 10 5 5.7 ± 1.1
0.5 ± 0.4
1.0 ± 0.3
0.6 ± 0.3
<0.6 ± 0.2
5.5 ± 1.6
3.9 ± 0.6
0.3 ± 0.1
4.3 ± 1.4
3.5 ± 0.4
<0.8 ± 0.2
1.0 ± 0.5
9.1, 11 24, 15 N 3 3 3 2 2 4 3 3 3 3 3 3 3 3
Topological 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 P1-pwt-tet P1-pwt-tet Cleavage Site none PstI Postcleavage Treatment amp r tet r /amp r x 10 5 15 ± 4.3
9.5, 11 N 11 2
Structural Requirements at the P Element 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.
Structural Requirements at the P Element DNA Termini
• 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.
Structural Requirements at the P
P1-Pwt-tet P1-Pwt-tet XB + 4 wt XB + 4 wt XB + 4 26 XB + 4 26 SacI wt SacI wt SacI wt SacI wt SacI 26 SacI 26 SacI 26 SacI 26 XB wt XB wt XB wt XB wt XB 26 XB 26 XB 26 XB 26 PCR wt PCR 26
Element DNA Termini
amp r tet r /amp r x 10 5 Donor Plasmid Cleavage site Postcleavage treatment SacI BsaI BsaI BsaI BsaI BsaI BsaI BsaI BsaI none PstI XbaI BsaI XbaI BsaI SacI SacI SacI SacI SacI SacI SacI T4 Dna polymerase CIP CIP, PNK 15 ± 4.3
9.5, 11 14, 12 0.6 ± 0.3
<0.3
<0.2, <0.2
46 ± 25 1.7 ± 0.4
77 ± 15 58 ± 13 T4 Dna polymerase CIP CIP, PNK Klenow + 4 dNTPs Klenow + 3dNTPs ddGTP 1.3 ± 0.7
<0.3, <0.3
0.9, 1.1
1.4, 3.5
<0.2 ± 0.1
2.4 ± 1.3
<0.2 ± 0.1
Klenow + dATP, dCTP, TTP <0.3, <0.3
<0.2 ± 0 Klenow + 4 dNTPs Klenow + 3dNTPs ddGTP Klenow + dATP, dCTP, TTP <0.3, 0.4
Klenow + 4 dNTPs 1.5 ± 0.3
Klenow + 4 dNTPs <0.5 ± 0.2
<0.3 ±0.2
<0.4 ± 0.1
N 6 6 7 2 2 11 2 2 3 1 2 10 6 2 5 10 4 2 4 7 4 2 4 4
Structure of the Transposition Products
• 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).
Structure of the Transposition Products
• 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.
Discussion
• Transposition depends on the addition of partially purified P Element Transposase
Discussion
• 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.
Discussion
• 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.
• Transposition was greatly diminished by the deletion of the transposase binding site at the 3’ terminus.
Discussion
• 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.
Discussion
• • 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.
Discussion
• 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.
Discussion
• Ideal conditions appear to be Transposase induced Wild Type Donor 30ºC temp SacI digest treated with CIP Run as long as possible With excess GTP
Discussion
• 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.