Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Peterson, Gregory C.; Reznikoff, William S.

doi:10.1074/jbc.m208968200

Cited by 24 publications

(19 citation statements)

References 17 publications

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“…Piv E59D and Piv D101E exhibit very low levels of recombination, and generating a DDE-, versus DED-, motif in the linear amino acid sequence of Piv (Piv E59D/D101E and Piv E59D/D104E) did not yield an active recombinase. These results are consistent with substitution analyses of the DDEmotif in Tn5 Tnp and the DEDD-motif in the Holliday junction resolvase RuvC (26,28). These studies showed that substitution of catalytic residues with nonacidic amino acids eliminates recombination activity, but interchanging aspartic acid and glutamic acid residues at catalytic positions (other than the first aspartic acid) is tolerated to give low levels of in vitro catalytic activity.…”

Section: Discussionsupporting

confidence: 78%

Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

et al. 2005

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Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine-or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.Piv catalyzes site-specific inversion of a 2.1-kb chromosomal segment encoding type 4 pilin genes of the human and cow eye pathogens Moraxella lacunata and Moraxella bovis, respectively (14,18,27). This DNA rearrangement results in phase variation of the type 4 pili (19) (Fig. 1). The invertible segment is bounded by identical 32-bp inverted repeats, invL and invR, where Piv mediates recombination. In addition, Piv interacts with an accessory site, sub1, which may facilitate assembly of an active synaptic complex (32).Piv is unique among prokaryotic site-specific DNA invertases because it does not belong to either the tyrosine-or serinerecombinase families. Instead, Piv shares significant amino acid identity and similarity with the transposases of insertion sequences (IS) from the IS110/IS492 family, including three highly conserved amino acid motifs: GDK, KTDDAA, and PSG (14, 24). Piv variants containing alanine or glycine substitutions at multiple positions within each motif are defective for mediating inversion but still bind to the inv and sub sites. The essential role of these conserved motifs in Piv catalysis of DNA inversion suggests that Piv and the transposases of the IS110/IS492 family (Piv/MooV family of DNA recombinases) use similar catalytic mechanisms (25, 31).Most identified transposases belong to the superfamily of retroviral integrases (IN) and are typified by a conserved DDE catalytic motif. The acidic residues of the DDE mot...

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Section: Discussionsupporting

confidence: 78%

Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

et al. 2005

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“…However, AGO2 and -3 differ from the other AGOs in that they have an additional aspartate as the third coordinating residues in place of the conserved histidine. This third carboxylic acid residue is not likely to impair Slicer activity because two other related enzymes, RNaseH1 and Tn5 integrase, can use aspartate efficiently in that position (13,59,60). Most of the AGO proteins, also like hAGO2, have a conserved histidine at the equivalent of AGO1 position 798, and it is likely that they selectively recruit siRNAs or a subset of miRNAs and use them as guides in RNA cleavage reactions.…”

Section: Selective Srna Recruitment and Slicer Activity In Other Arabmentioning

confidence: 99%

Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs

Baumberger

Baulcombe²

2005

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

933

795

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ARGONAUTE (AGO) RNA-binding proteins are involved in RNA silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced silencing complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA-and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNAsilencing pathways. Here, we show that AGO1 selectively recruits certain classes of short silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgenederived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced silencing complex. The Slicer activity fractionates as a complex of Ϸ150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.posttranscriptional regulation ͉ ribonuclease ͉ viral RNA ͉ silencing A RGONAUTE (AGO) proteins are implicated in RNAsilencing processes that also involve 21-to 26-nt short RNAs (sRNAs) (1) cleaved from double-stranded or partially doublestranded (ds) RNAs by the RNase III enzyme Dicer. There are several types of RNA-silencing mechanisms, including RNA interference (RNAi), the micro RNA (miRNA) pathway, and RNAdirected chromatin silencing (1). RNAi is a type of RNA silencing in which the Dicer substrate is fully double stranded, the sRNA cleavage product is short interfering RNA (siRNA), and the outcome is targeted destruction of siRNA-complementary RNAs. The miRNA pathway is similar except that the Dicer substrate is an inverted repeat RNA with a partially ds structure, the sRNA is referred to as a miRNA, and the target RNAs can be suppressed at the translational level or degraded as in RNAi, depending on the degree of complementarity between the sRNA and its target. Plants possess an additional class of degradative sRNAs called transacting siRNAs (ta-siRNAs) whose formation depends on the miRNAmediated cleavage of their precursor and its conversion into a dsRNA by RDR6 (2-4). The last pathway, RNA-directed chromatin silencing, is similar to RNAi, but the siRNA targets are either DNA or chromatin-associated RNAs and the outcome is DNA methylation or histone modification at the target locus.In the best understood of these RNA-silencing mechanisms, the duplex siRNAs or miRNAs produced by Dicer are unwound in an ATP-dependent process. One ...

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“…The published cocrystal structure predicts the existence of five different Tnp regions that make ES contacts; two regions form cis contacts, and three regions form trans contacts (9). Three of these regions have been extensively studied in other work (2,5,16,17,19,25,27,30,31). In this communication we present analyses of the remaining two regions (the cis contact region from residue 342 to 348 and the trans contact region from residue 438 to 445) and verify, through genetics and biochemistry, that these regions play an important role in synaptic complex formation.…”

Section: Discussionmentioning

confidence: 99%

Site-Directed Mutagenesis Studies of Tn 5 Transposase Residues Involved in Synaptic Complex Formation

2007

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Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.Transposition is the movement of a defined DNA segment, a transposon, from one location to another within the same or different DNA molecules. Tn5, which is a 5.8-kbp prokaryotic transposon, consists of two inverted terminal repeats, IS50L and IS50R, that flank the genes encoding for antibiotic resistance ( Fig. 1A) (5). IS50R carries the gene for Tn5 transposase (Tnp), the only protein catalyzing Tn5 transposition steps (13). Each IS50 element is flanked by two inverted 19-bp end sequences (ESs) (12), called the outside end (OE) and the inside end (IE), that are recognized by Tnp during the transposition process ( Fig. 1) (4). During transposition, the transposon is completely excised from donor DNA and inserted into a target DNA. This movement is carried out through a conservative "cut-and-paste" mechanism (8), which is explained and shown schematically in Fig. 2. The transposition process is mediated by three factors: Tnp, a transposon (which is bracketed by two inverted ESs), and Mg 2ϩ ions. Tnp is critical in many transposition steps, including binding to the transposon DNA ESs (24), bringing the two ESs together via formation of a protein homodimer that results in synaptic complex formation, cleaving the DNA adjacent to the ESs, and inserting the transposon into a target DNA (3,7,8,(20)(21)(22)(23).The synaptic complex is a key intermediate in Tn5 transposition. This is because all catalysis occurs in trans, with the Tnp monomer that initially bound to one ES performing the catalytic events on the ES-donor backbone (DBB) border that was initially bound by the other Tnp monomer. Throughout our studies we used a previously described hyperactive mutant (E54K, M56A, L372P) of Tnp, called EK/LP, as a control (9,11,21,29,31). The previously published cocrystal structure of EK/LP bound to the OE ES (Fig. 3) (generated from PDB ID 1MUH) predicts the existence of multiple Tnp-ES DNA contacts both in cis and in trans (9). These contacts are presumed to play key roles in stabilizing the synaptic complex and in establishing de...

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Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Cited by 24 publications

References 17 publications

Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs

Site-Directed Mutagenesis Studies of Tn 5 Transposase Residues Involved in Synaptic Complex Formation

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