Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3757-3765.1992 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3757 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3757-3765 ◽

Cited By ~ 31

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.

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Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3167 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3167-3175 ◽

Cited By ~ 41

Author(s):

G Pan ◽

K Luetke ◽

P D Sadowski

Keyword(s):

Amino Acid ◽

Family Members ◽

Amino Acid Sequences ◽

Chemical Mechanism ◽

Complementation Analysis ◽

Phosphodiester Bond ◽

Flp Recombinase ◽

Group I ◽

In Cis

The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

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Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3167-3175.1993 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3167-3175

Author(s):

G Pan ◽

K Luetke ◽

P D Sadowski

Keyword(s):

Amino Acid ◽

Family Members ◽

Amino Acid Sequences ◽

Chemical Mechanism ◽

Complementation Analysis ◽

Phosphodiester Bond ◽

Flp Recombinase ◽

Group I ◽

In Cis

The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

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Structure-Function Analysis of IntDOT

Journal of Bacteriology ◽

10.1128/jb.01052-09 ◽

2009 ◽

Vol 192 (2) ◽

pp. 575-586 ◽

Cited By ~ 5

Author(s):

Seyeun Kim ◽

Brian M. Swalla ◽

Jeffrey F. Gardner

Keyword(s):

Homology Modeling ◽

Protein Interactions ◽

Active Site ◽

Dna Cleavage ◽

Function Analysis ◽

Indicator Strain ◽

Amino Terminal ◽

Dna Ligation ◽

Polypeptide Backbone

ABSTRACT CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

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In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽

10.1093/genetics/149.4.1649 ◽

1998 ◽

Vol 149 (4) ◽

pp. 1649-1663

Author(s):

Oliver Z Nanassy ◽

Kelly T Hughes

Keyword(s):

Amino Acids ◽

Biochemical Analysis ◽

Mutational Analysis ◽

Recombination Reaction ◽

Recombination Site ◽

Dna Inversion ◽

Hin Recombinase ◽

One Step ◽

A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

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Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714025188 ◽

2015 ◽

Vol 71 (2) ◽

pp. 256-265 ◽

Cited By ~ 1

Author(s):

Takashi Kawamura ◽

Tomoki Kobayashi ◽

Nobuhisa Watanabe

Keyword(s):

Electron Density ◽

Active Site ◽

Dna Cleavage ◽

Metal Ion ◽

Manganese Ions ◽

Metal Ion Binding ◽

Time Resolved ◽

Manganese Ion ◽

Ion Binding Sites ◽

Metal Ion Binding Sites

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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