scholarly journals The mechanism of sequence-specific DNA cleavage and strand transfer by phi X174 gene A* protein.

1993 ◽  
Vol 268 (32) ◽  
pp. 23830-23836
Author(s):  
R Hanai ◽  
J.C. Wang
Keyword(s):  
Dna Cleavage ◽  
Strand Transfer

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

1992 ◽  
Vol 12 (9) ◽  
pp. 3757-3765
Author(s):  
J W Chen ◽  
B R Evans ◽  
S H Yang ◽  
H Araki ◽  
Y Oshima ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Substrate Binding ◽  
Point Mutations ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Site Specific ◽  
Arginine Residues ◽  
Active Site Tyrosine ◽  
Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

scholarly journals Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly

2017 ◽  
Vol 114 (5) ◽  
pp. E669-E678 ◽  
Author(s):  
Emilien Nicolas ◽  
Cédric A. Oger ◽  
Nathan Nguyen ◽  
Michaël Lambin ◽  
Amandine Draime ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Initial Step ◽  
Strand Transfer ◽  
Wild Type ◽  
Multiresistant Pathogens ◽  
Activated State ◽  
Transpososome Assembly

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

HIV-1 DNA integration: Mechanism of viral DNA cleavage and DNA strand transfer

Cell ◽  
1991 ◽  
Vol 67 (6) ◽  
pp. 1211-1221 ◽  
Author(s):  
Alan Engelman ◽  
Kiyoshi Mizuuchi ◽  
Robert Craigie
Keyword(s):  
Dna Cleavage ◽  
Strand Transfer ◽  
Viral Dna ◽  
Dna Integration ◽  
Integration Mechanism ◽  
Dna Strand ◽  
Hiv 1

scholarly journals Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

1992 ◽  
Vol 12 (9) ◽  
pp. 3757-3765 ◽  
Author(s):  
J W Chen ◽  
B R Evans ◽  
S H Yang ◽  
H Araki ◽  
Y Oshima ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Substrate Binding ◽  
Point Mutations ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Site Specific ◽  
Arginine Residues ◽  
Active Site Tyrosine ◽  
Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

1999 Roche Diagnostics Prize for Biomolecular and Cellular Research / Prix Roche Diagnostics 1999 pour la recherche en biologie moléculaire et cellulaireStudies on a "jumping gene machine": Higher-order nucleoprotein complexes in Mu DNA transposition

1999 ◽  
Vol 77 (6) ◽  
pp. 487-492 ◽  
Author(s):  
George Chaconas
Keyword(s):  
Dna Cleavage ◽  
Higher Order ◽  
Strand Transfer ◽  
Dna Transposition ◽  
Oh Groups ◽  
Dna Breakage ◽  
Gradual Process ◽  
Nucleoprotein Complexes ◽  
Roche Diagnostics

Studies in my lab have focused on DNA transposition in the bacterial virus, Mu. In vitro studies have shown that Mu DNA transposition is a three-step process involving DNA breakage, strand transfer and DNA replication. In the first step, a nick is introduced at each end of the transposon. The liberated 3'-OH groups subsequently attack a target DNA molecule resulting in strand transfer. The transposon DNA, now covalently linked to the target, is finally replicated to generate the transposition end-product, referred to as a cointegrate. The DNA cleavage and strand transfer reactions are mediated by a "jumping gene machine" or transpososomes, which we discovered in 1987. They are assembled by bringing together three different DNA regions via a process involving multiple protein-DNA and protein-protein interactions. The action of four different proteins is required in addition to protein-induced DNA bending or wrapping to overcome the intrinsic stiffness of DNA, which would ordinarily prohibit the assembly of such a structure. Transpososome assembly is a gradual process involving multiple steps with an inherent flexibility whereby alternate pathways can be used in the assembly process, biasing the reaction towards completion under different conditions.Key words: DNA transposition, transposons, higher-order nucleoprotein complexes, DNA breakage and reunion, site-specific recombination.

scholarly journals Mu Transpositional Recombination: Donor DNA Cleavage and Strand Transfer in trans by the Mu Transposase

Cell ◽  
1996 ◽  
Vol 85 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Harri Savilahti ◽  
Kiyoshi Mizuuchi
Keyword(s):  
Dna Cleavage ◽  
Strand Transfer ◽  
In Trans

scholarly journals A Bacterial TrwC Relaxase Domain Contains a Thermally Stable α-Helical Core

2003 ◽  
Vol 185 (14) ◽  
pp. 4226-4232 ◽  
Author(s):  
José-Luis R. Arrondo ◽  
Izaskun Echabe ◽  
Ibón Iloro ◽  
Miguel-Ángel Hernando ◽  
Fernando de la Cruz ◽  
...  
Keyword(s):  
Outer Layer ◽  
Dna Cleavage ◽  
Correlation Spectroscopy ◽  
Slow Heating ◽  
Strand Transfer ◽  
Fast Heating ◽  
Heating And Cooling ◽  
State Process ◽  
A Cell ◽  
Α Helix

ABSTRACT The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. The TrwC-N275 fragment comprises the 275-amino-acid N-terminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain). It can be easily purified by keeping a cell lysate at 90°C for 10 min. Infrared spectroscopy shows that this domain has a predominantly α/β structure with some amount of unordered structure. Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation. The denaturation temperature is increased in the protein after the fast-heating thermal shock. Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation. After aggregation, the α-helix percentage is not altered and α-helix signal does not show in the correlation maps, meaning that the helices are not affected by heating. The results indicate that the domain has an α-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of β-sheet and unordered structure that aggregates under slow heating. The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding.

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