Evidence for “Unseen” Transposase–DNA Contacts

Steiniger-White, Mindy; Bhasin, Archna; Lovell, Scott; Rayment, Ivan; Reznikoff, William S.

doi:10.1016/s0022-2836(02)00877-x

Cited by 19 publications

(23 citation statements)

References 24 publications

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“…On the basis of the published partial X-ray IN structures 1EX4 (37) and 1K6Y (4), a 3D IN model was assembled by using the Brugel package. The two Mg 2ϩ ions and the viral DNA were added according to the crystal structure of the structurally homologous Tn5 transposase crystal structure 1MM8 (33). Host DNA was added to the structure based on available contacts with the viral DNA-IN complex.…”

Section: Methodsmentioning

confidence: 99%

Mutations in Human Immunodeficiency Virus Type 1 Integrase Confer Resistance to the Naphthyridine L-870,810 and Cross-Resistance to the Clinical Trial Drug GS-9137

Hombrouck

Voet

Remoortel

et al. 2008

Antimicrob Agents Chemother

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Section: Methodsmentioning

confidence: 99%

Mutations in Human Immunodeficiency Virus Type 1 Integrase Confer Resistance to the Naphthyridine L-870,810 and Cross-Resistance to the Clinical Trial Drug GS-9137

Hombrouck

Voet

Remoortel

et al. 2008

Antimicrob Agents Chemother

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“…Previous co-crystal structures of Tn5 Tnp paired ends complexes (PECs) show a β-loop (amino acids 240-260) of Tnp contacting nucleotide pairs 3-5 of the recognition end sequence (ES) (13,15,16). See Figure 2.…”

Section: In Vivo Analysis Of β-Loop Mutantsmentioning

confidence: 98%

“…The hyperactivity was shown to stem from the orientation of the adenine-thymine (A-T) bp at position 4 (15). When the T was located on the non-transferred strand (see Figure 2) closest to the β-loop, as in the ME, hyperactivity resulted.…”

mentioning

confidence: 99%

Mutation of Tn5 Transposase β-Loop Residues Affects All Steps of Tn5 Transposition: The Role of Conformational Changes in Tn5 Transposition

Steiniger¹,

Metzler²,

Reznikoff³

2006

Biochemistry

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X-ray co-crystal structures of Tn5 transposase (Tnp) bound to its 19 base pair (bp) recognition end sequence (ES) reveal contacts between a β-loop (amino acids 240-260) and positions 3, 4, 5 and 6 of the ES. Here we show that mutations of residues in this loop affect both in vivo and in vitro transposition. Most mutations are detrimental, while some mutations at position 242 cause hyperactivity. More specifically, mutations to the β-loop affect every individual step of transposition tested. Mutants performing in vivo and in vitro transposition less efficiently also form fewer synaptic complexes, while hyperactive Tnps form more synaptic complexes. Surprisingly, two hypoactive mutations, K244R and R253L, also affect the cleavage steps of transposition with a much more dramatic effect on the second double end break (DEB) complex formation step, indicating that the β-loop likely plays an important roll in positioning the substrate DNA within the catalytic site. Finally, all mutants tested decrease efficiency of the final transposition step, strand transfer. A disparity in in vitro cleavage rate constants for mutants with changes to the proline at position 242 on transposons flanked by ESes differing in the orientation of the A-T base pair at position 4 allows us to postulate that P242 contacts the position 4 nucleotide pair. Based on these data, we propose a sequential model for end cleavage in Tn5 transposition in which the uncleaved PEC is not symmetrical and conformational changes are necessary between the first and second cleavage events and also for the final strand transfer step of transposition.Transposition is the process of moving DNA from one location to another. Tn5 is a well-studied composite, prokaryotic transposon consisting of two insertion sequences, IS50R and IS50L flanked by 19 base pair (bp) inverted repeats termed outside ends (OEs) (1). IS50R encodes the transposase (Tnp), the only protein required for transposition in this system (2). Tn5 is mobilized using a cut-and-paste mechanism in which the transposon is completely removed from its original location before being inserted in a new DNA site (3).Following translation, Tnp initially interacts with DNA non-specifically, but then localizes the OE by both inter and intramolecular transfer, possibly via a looping mechanism ( Figure 1A, C. D. Adams, personal communication and (4)). The OE bound Tnps then form a dimeric synaptic complex, the nucleoprotein complex required for catalysis (5-7). Following synapsis, cleavage occurs at one OE/flanking donor backbone (dbb) DNA junction (+1) followed by the other. First, a water molecule activated by Mg 2+ attacks the phosphodiester backbone of one DNA strand at the +1 location resulting in the generation of a 3′ hydroxyl group ( Figure 1B, column 1). This 3′ hydroxyl group then attacks the opposite DNA strand creating a hairpin intermediate and releasing the dbb DNA (8). The synaptic complex containing one cleaved * To whom correspondence should be addressed: William S. Reznikoff, Tel: (608) F...

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“…The active site residues (sticks) are located within the PIWI domain. b) A comparison of the RNase Hlike fold for Tn5 Transposase [58], PfAgo PIWI domain [10], and E. coli RNase HI [59]. The equivalent secondary structural elements are presented in identical colors for each structure.…”

Section: New Classes Of Small Rnas Distribute Into Distinct Argonautementioning

confidence: 99%

Argonautes confront new small RNAs

Faehnle

Joshua‐Tor

2007

Current Opinion in Chemical Biology

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Argonaute is at the heart of all effector complexes in RNA interference. In the classical RNAi pathway Argonaute functions as the Slicer enzyme that cleaves a mRNA target directed by a complementary siRNA. Two recently described Argonaute protein subfamilies mediate distinct functions in RNAi. The Piwi subfamily functions in the germline through a novel class of small RNAs that are longer than Argonaute specific si-and miRNAs. Piwi-interacting RNAs (piRNAs) carry a 2' O-methylation on their 3' end and appear to be synthesized by a Piwi Slicer dependent mechanism. Piwi/piRNA complexes in mammals and flies are directly linked to the control of transposable elements during germline development. Amplified RNAi in C. elegans is mediated by secondary siRNAs selectively bound to secondary Argonautes (SAGOs) that belong to a worm specific Argonaute subfamily (WAGO). Secondary siRNAs are 5' triphosphorylated which may allow specific loading into SAGO complexes that are rate limiting for RNAi in C. elegans. Interestingly SAGOs lack conserved Slicer amino acid residues and probably act in a Slicer-independent fashion.

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Evidence for “Unseen” Transposase–DNA Contacts

Cited by 19 publications

References 24 publications

Mutations in Human Immunodeficiency Virus Type 1 Integrase Confer Resistance to the Naphthyridine L-870,810 and Cross-Resistance to the Clinical Trial Drug GS-9137

Mutations in Human Immunodeficiency Virus Type 1 Integrase Confer Resistance to the Naphthyridine L-870,810 and Cross-Resistance to the Clinical Trial Drug GS-9137

Mutation of Tn5 Transposase β-Loop Residues Affects All Steps of Tn5 Transposition: The Role of Conformational Changes in Tn5 Transposition

Argonautes confront new small RNAs

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