Half-site strand transfer by step-arrest mutants of yeast site-specific recombinase Flp

Serre, Marie-Claude; Jayaram, Makkuni

doi:10.1016/0022-2836(92)90391-v

Cited by 26 publications

(22 citation statements)

References 11 publications

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“…The His-305 variant of Flp has previously been found to cleave full sites (with no strand discontinuity) efficiently and to accumulate the cleavage product (25). However, it fails to cleave a half-site or cleaves it extremely inefficiently (26). The weaker cleavage by Flp(H305L) than Flp observed here may be due to the strand discontinuity in the substrates tested.…”

Section: Formation Of Covalent Flp-dna Complexes In Activated and Noncontrasting

confidence: 50%

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

Knudsen

Lee

Lisby

et al. 1998

Journal of Biological Chemistry

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The strand joining step of recombination mediated by the Flp site-specific recombinase involves the attack of a 3-phosphotyrosyl bond by a 5-hydroxyl group from DNA. The nucleophile in this reaction, the 5-OH, can be substituted by glycerol or other polyhydric alcohols. The strand joining and glycerolysis reactions are mechanistically equivalent and are competitive to each other. The target diester in strand joining can be a 3-phosphate covalently linked either to a short tyrosyl peptide or to the whole Flp protein via Tyr-343. By contrast, only the latter type of 3-phosphotyrosyl linkage is a substrate for glycerolysis. As a result, in activated DNA substrates (containing the scissile phosphate linked to a short The Flp site-specific recombinase of Saccharomyces cerevisiae is a member of the integrase family of recombinase proteins (2, 3). These proteins carry out recombination "conservatively," i.e. without DNA degradation or synthesis, and in the absence of exogenous energy input. This "break-join" mechanism follows from the ability of integrase-type recombinases to carry out two types of transesterification reactions, first to break the parental DNA strand and then to form the recombinant strand. The cleavage and joining steps of recombination follow a basic topoisomerase I strategy. Strand cleavage is mediated by an active site tyrosine residue, Tyr-343 in Flp. As a result, a 3Ј-phosphotyrosine bond and a 5Ј-hydroxyl group are formed at either side of the nick. Strand joining in the recombinant mode is chemically a reversal of the cleavage step, except that the reaction occurs between the broken strands of two DNA partners.Several interesting features of the cleavage and joining steps of the Flp reaction have been revealed (4 -7). The active Flp entity for strand cutting is a dimer (8, 9). Within the dimer, one Flp monomer, bound adjacent to a scissile phosphodiester bond, orients it for attack by Tyr-343 of the second Flp monomer bound across the strand exchange region (or spacer). Thus, the target phosphate is oriented in cis, while the cleavage nucleophile is donated in trans. The strand exchange reaction also follows the "cis-orientation/trans-nucleophilic attack" paradigm. In this case, the target phosphate belongs to the 3Ј-phosphotyrosine bridge, and the nucleophile is the 5Ј-hydroxyl group formed on the partner DNA. Once the target has been activated by Flp, a variety of exogenous nucleophiles can mimic the action of the native ones. Thus, strand breakage can be effected by tyramine, phenol, or hydrogen peroxide (6). Similarly, the 3Ј-phosphotyrosyl bond formed by Flp cleavage can be attacked by glycerol or polyhydric alcohols, by water (or the hydroxide ion), or even by a vicinyl 2Ј-hydroxyl group in a DNA-RNA hybrid substrate (10, 11).A simplified form of the recombination reaction (that involves the breakage and reformation of only one phosphodiester bond) can be performed using suitably designed "halfsite" substrates (12, 13). These substrates were fashioned after the half-sites employed for the...

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Section: Formation Of Covalent Flp-dna Complexes In Activated and Noncontrasting

confidence: 50%

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

Knudsen

Lee

Lisby

et al. 1998

Journal of Biological Chemistry

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“…It is possible that the absence of His-305, combined with the mismatched spacer configuration, perturbs the normal protein interactions that lead to catalysis. The histidine variants are also known to test as cleavage incompetent when provided with half-site substrates (23). When a triad arginine variant of Flp was mixed with Flp(Y343F), they complemented each other, as evidenced by the cleavage detected within the bubbled full site (lanes 5 and 9 in Fig.…”

Section: Resultsmentioning

confidence: 99%

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

1994

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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 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.The Flp protein of Saccharomyces cerevisiae is a conservative, site-specific DNA recombinase that belongs to the Int (A integrase) family of recombinases (1, 3). Members of this family execute recombination in two sequential steps. The first pair of strand cleavage-joining reactions produces a Holliday intermediate which, following branch migration, is resolved into recombinants by the second pair of cleavage-joining reactions. The Int family recombinases use an active-site tyrosine as the nucleophile to attack the scissile phosphodiester during the strand breakage step. In Flp, this tyrosine residue is Tyr-343 (9). The active-site tyrosine is one of the invariant tetrad residues of the Int family (1, 3). The other three invariant residues are two arginines and a histidine (the RHR triad; The active-site configuration of two Int family members, Flp and the Zygosaccharomyces rouxii recombinase R, inferred from recombination reactions containing half-site substrates and step arrest mutants of the recombinases suggests potential solutions to the problems addressed above (6,7,13,17,24). A monomer of Flp or R harbors a partial active site; a complete active site is assembled by contribution of residues from more than one recombinase monomer. In the shared active site, three of the invariant Int family residues, the RHR triad (Arg-191, His-305, and Arg-308 in Flp; Arg-207, His-317, and Arg-320 in R), are derived from one monomer; the active-site tyrosine (Tyr-343 in Flp and Tyr-358 in R) is provided by a second mon...

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“…Strikingly, mutants that retained a low DNA relaxation activity were deficient in covalent complex formation, suggesting that the cleavage of a small synthetic substrate may be a more stringent assay than the relaxation assay. Indeed, it was shown previously for the Flp recombinase that some mutants showing activity on large substrates were deficient in cleavage activity when tested on small synthetic substrates (40).…”

Section: Most Int Ssv Mutants Are Impaired In Covalent Complex Formatmentioning

confidence: 98%

Mutational Analysis of the Archaeal Tyrosine Recombinase SSV1 Integrase Suggests a Mechanism of DNA Cleavage in trans

Letzelter¹,

Duguet

Serre

2004

Journal of Biological Chemistry

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The only tyrosine recombinase so far studied in archaea, the SSV1 integrase, harbors several changes in the canonical residues forming the catalytic pocket of this family of recombinases. This raised the possibility of a different mechanism for archaeal tyrosine recombinase. The residues of Int SSV tentatively involved in catalysis were modified by site-directed mutagenesis, and the properties of the corresponding mutants were studied. The results show that all of the targeted residues are important for activity, suggesting that the archaeal integrase uses a mechanism similar to that of bacterial or eukaryotic tyrosine recombinases. In addition, we show that Int SSV exhibits a type IB topoisomerase activity because it is able to relax both positive and negative supercoils. Interestingly, in vitro complementation experiments between the inactive integrase mutant Y314F and all other inactive mutants restore in all cases enzymatic activity. This suggests that, as for the yeast Flp recombinase, the active site is assembled by the interaction of the tyrosine from one monomer with the other residues from another monomer. The shared active site paradigm of the eukaryotic Flp protein may therefore be extended to the archaeal tyrosine recombinase Int SSV .Tyrosine recombinases form a large family of site-specific recombinases comprising more than 150 members, most of which were identified on the basis of sequence similarities (1, 2). Within this family, several subfamilies can be defined such as the -phage integrase family, the Xer recombinases family, or the yeast plasmid recombinases family (1). The hallmark of tyrosine recombinases is the conservation of six noncontiguous residues: Arg I , Lys ␤ , His II , Arg II , His/Trp, Tyr (Table I). This motif is directly involved in catalysis of DNA strand cleavage and strand exchange (for review, see Ref.3). Five of the six residues are located within the highly conserved boxes I and II found in tyrosine recombinases (1, 4, 5), whereas the sixth residue, Lys ␤ , was identified by alignments with the eukaryotic topoisomerases IB (6). Two different structural organizations of this motif have been described from crystallographic data. In prokaryotic tyrosine recombinases XerD (7), Cre (8), HP1 integrase (9), and -Int (10, 11), the six active site residues come from a single monomer, whereas the eukaryotic Flp recombinase presents a shared active site, where the catalytic tyrosine is provided by one monomer, and the five other residues are from another monomer (12). In this latter case, the active site is created by dimer association. As a consequence of this organization, Flp realizes trans cleavage (13, 14), whereas prokaryotic recombinases act in cis (8,9,(15)(16)(17). Cis cleavage is the result of cis activation/cis cleavage where the tyrosine of the bound monomer attacks the nearby activated phosphate. In trans cleavage, binding of a monomer to its site leads to activation of the adjacent phosphodiester that will be attacked by a nucleophile (here a tyrosine) provided in tran...

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Half-site strand transfer by step-arrest mutants of yeast site-specific recombinase Flp

Cited by 26 publications

References 11 publications

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

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

Mutational Analysis of the Archaeal Tyrosine Recombinase SSV1 Integrase Suggests a Mechanism of DNA Cleavage in trans

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