Guanosine triphosphate acts as a cofactor to promote assembly of initial P-element transposase–DNA synaptic complexes

Tang, Mei; Cecconi, Ciro; Kim, Helen; Bustamante, Carlos; Rio, Donald C.

doi:10.1101/gad.1317605

Cited by 25 publications

(58 citation statements)

References 34 publications

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“…The P-element transposase catalyzes DNA cleavage within the 31-bp TIRs to create 17-nt 3′ single-strand extensions at both the donor site and the transposon ends (14,15). The cleaved P-element ends remain tightly bound by the transposase enzyme (10,16). The flanking donor site DNA is released after P-element cleavage (16) and needs to be efficiently repaired by endogenous DNA repair mechanisms.…”

Section: Irbp Complexmentioning

confidence: 99%

“…The Drosophila P-element transposon encodes a GTP-dependent site-specific DNA transposase/integrase family enzyme (9,10). At each end of the P-element transposon are perfect 31-bp terminal inverted repeats (TIRs), 11-bp internal inverted repeats that serve as enhancers of transposition, and internal 10-bp transposase binding sites (11)(12)(13) (Fig.…”

Section: Irbp Complexmentioning

confidence: 99%

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Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

Francis

Roche

Cho

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed "Inverted Repeat Binding Protein (IRBP) 18" and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.T ransposable elements contribute significantly to the organization and evolution of all eukaryotic genomes. Recent estimates of transposon content within the Drosophila melanogaster genome are between 5% and 10%, and in humans over half the genome is composed of mobile elements (1, 2). Although many of these elements, including the Drosophila P-element transposon, are still active (3), the cellular mechanisms used to combat the genotoxic effects of DNA double-strand breaks (DSBs) generated by transpositional recombination are not fully understood. The Drosophila P-transposable element provides an excellent model for understanding the ancient mechanisms used by the cell to counteract newly invading parasitic mobile DNA elements (4).The P-element transposon is a mobile DNA element that spread through wild populations of D. melangaster ∼100 y ago after most common laboratory strains were isolated (5, 6). P elements were identified by studying a genetic syndrome called "P-M hybrid dysgenesis." It was observed that males from wild populations (P strains) crossed to females from isolated laboratory stocks (M strains) yielded progeny that had germline mutations, temperature-sensitive sterility, and atypical male recombination (6). Reciprocal crosses yielded phenotypically normal progeny. The P element was shown to be the causative agent of these so-called P-M hybrid dysgenesis phenotypes by molecular analyses showing that P elements were present in variable locations in P strains yet totally absent from most M strains (7,8).The Drosophila P-element transposon encodes a GTP-dependent site-specific DNA transposase/integrase family enzyme (9, 10). At each end of the P-element transposon are perfect 31-bp terminal inverted repeats (TIRs), 11-bp internal inverted repeats that serve as enhancers of transposition, and internal 10-bp transposase binding sites (11-13) (Fig. 1A). The P-element transposase catalyzes DNA cleavage within the 31-bp TIRs to create 17-nt 3′ single-strand extensions at both the donor site and the transposon ends (14, 15)...

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Section: Irbp Complexmentioning

confidence: 99%

Section: Irbp Complexmentioning

confidence: 99%

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

Francis

Roche

Cho

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Although divalent metal ions are universally required cofactors for transposase proteins (1,12,13), the P element reaction is unique among this family of polynucleotidyltransferases in the use of GTP as a cofactor (9). Recent studies have shown that GTP promotes pre-cleavage synaptic complex assembly between the P element termini and the transposase protein (14). Previous in vitro studies had also shown that nonhydrolyzable GTP analogs support both the P element DNA cleavage and joining reactions (9).…”

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confidence: 99%

Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition

Tang

Cecconi

Bustamante

et al. 2007

Journal of Biological Chemistry

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P elements are a family of transposable elements found inDrosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.Mobile genetic elements are ubiquitous among both prokaryotic and eukaryotic organisms (1). Genome sequencing projects have shown that transposable elements make up a substantial fraction of eukaryotic genomes, including 49% of the human genome (2, 3). These mobile elements can lead to mutations and genome rearrangements and appear to play a role in genome evolution (4, 5). The mechanisms by which transposons are mobilized can be grouped based upon whether there is a DNA or an RNA intermediate (1,4,5). P elements use a cut-and-paste mechanism with a DNA intermediate, related to those used by the Tn5, Tn10, and Tn7 prokaryotic transposons and the eukaryotic Tc1/mariner family (6 -8). Studies of P element transposition in vitro have demonstrated cofactor requirements for both GTP and magnesium ions (Mg 2ϩ ) (6, 9 -11). Although divalent metal ions are universally required cofactors for transposase proteins (1, 12, 13), the P element reaction is unique among this family of polynucleotidyltransferases in the use of GTP as a cofactor (9). Recent studies have shown that GTP promotes pre-cleavage synaptic complex assembly between the P element termini and the transposase protein (14). Previous in vitro studies had also shown that nonhydrolyzable GTP analogs support both the P element DNA cleavage and joining reactions (9). Furthermore, GTP does not effect the binding of P element transposase to the high affinity sites near the transposon termini, as assayed by DNase I footprinting (9, 15). Whereas some transposon systems have been studied with respect to the assembly state of their transposase, the oligomeric state of the active P element transposase protein and how it initially interacts with P element DNA are unknown. In cases where it has been studied, other transpos...

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“…To explain frequent local insertion (Zhang and Spradling 1993), proteins bound to P-element termini (Lee et al 1996;Beall and Rio 1998;Tang et al 2005) are thought to tether the excised element to the unexcised copy on the sister chromatid, giving rise to a local insertion event near the starting element. An alternative explanation is that local transposition may occur shortly after DNA replication, during which the two copies of the P elements are still in a close association.…”

Section: Resultsmentioning

confidence: 99%

Coincidence of P-Insertion Sites and Breakpoints of Deletions Induced by Activating P Elements in Drosophila

Sudi¹,

Zhang²,

Intrieri

et al. 2008

Genetics

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We isolated a set of seven deletions in the 67B region by activating a nearby P-element insertion. The structures of the deletions were characterized by cloning and sequencing. The results showed that the P-induced deletions occurred nonrandomly in the genomic sites. One breakpoint of the deletions was located precisely at the end of the starting element, i.e., at the end of the inverted terminal repeats. The other breakpoint was nearby the retained starting element and coincided with preferential P-element insertion sites that harbor transcription initiation activities. It is known that P elements induce male recombination near the starting elements, giving rise to deletions with one breakpoint precisely located at an inverted terminal repeat of the retained starting element. Database analyses further revealed that deletions generated in P-induced male recombination also contained the other breakpoint in genomic regions that coincided with preferential P-insertion sites. The results suggest that nonrandom distribution of the deletion breakpoints is characteristic of the mechanism by which P elements induce deletions near the starting elements.

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Guanosine triphosphate acts as a cofactor to promote assembly of initial P-element transposase–DNA synaptic complexes

Cited by 25 publications

References 34 publications

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition

Coincidence of P-Insertion Sites and Breakpoints of Deletions Induced by Activating P Elements in Drosophila

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