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M Jayaram

Publications and source records attributed to M Jayaram.

At least 37 records · Page 2Linked to original sources
PubMed1998

Alcoholysis and strand joining by the Flp site-specific recombinase. Mechanistically equivalent reactions mediated by distinct catalytic configurations.

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 Flp peptide), Flp(Y343F) can mediate the joining reaction utilizing the 5'-hydroxyl attack but fails to promote glycerolysis. Wild type Flp promotes both reactions in these substrates. The strand joining and glycerolysis reactions are absolutely dependent on the catalytic histidine at position 305 of Flp. Our results fit into a model in which a Flp dimer, with one monomer covalently attached to the 3'-phosphate, is essential for orienting the target diester or the nucleophile (or both) during glycerolysis. The requirement for this dimeric complex is relaxed in the strand joining reaction because of the ability of DNA to orient the nucleophile (5'-OH) by complementary base pairing. The experimental outcomes described here have parallels to the "cleavage-dependent ligation" carried out by a catalytic variant of Flp, Flp(R308K) (Zhu, X.-D., and Sadowski, P. D. (1995) J. Biol. Chem. 270, 23044-23054).

Alcohols↗
PubMed1998

Mutational analysis of domain II beta of bacteriophage Mu transposase: domains II alpha and II beta belong to different catalytic complementation groups.

This study examines the contribution of domain II beta of bacteriophage Mu transposase (A protein), a subdomain of the central catalytic domain II, to the transposition reaction. The properties of several point mutations implicate a role for this domain in facilitating metal-assisted assembly of the synaptic complex, as well as in intramolecular DNA strand transfer. Point mutations as well as deletions in domain II beta can be complemented by those in domain II alpha but not those in domain III alpha. Thus, residues within subdomains II alpha and II beta belong to different catalytic complementation groups.

Bacteriophage mu↗
PubMed1998

Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase.

Binding of a cleavage-incompetent mutant of the Flp recombinase induces a roughly square-planar geometry in synthetic immobile Holliday junctions. The branch points, which are rigidly fixed in these junctions in their free forms, tend to be more flexible in their protein-bound forms. Our results (1) suggest a plausible mechanism for the switching of the recombination complex from the Holliday-forming mode to the Holliday-resolving mode, (2) provide a rationale for previous observations that Flp resolves preformed immobile Holliday structures in the parental or in the recombinant mode in a relatively unbiased manner, and (3) accommodate two modes of DNA cleavage by Flp (transhorizontal or transdiagonal) in Holliday substrates.

Bacterial Outer Membrane Proteins↗
PubMed1998

Unveiling two distinct ribonuclease activities and a topoisomerase activity in a site-specific DNA recombinase.

The site-specific DNA recombinase Flp shows two types of RNA cleavage activities on hybrid DNA-RNA substrates. One targets the phosphodiester position involved in DNA recombination and follows a related mechanistic path. In this two-step reaction, first-strand scission is mediated by a nucleophilic attack of the scissile phosphodiester bond by the active site tyrosine of Flp. The resultant 3'-O-phosphoryl tyrosine bond is then attacked by the adjacent 2'-hydroxyl group. The second activity targets the immediately adjacent phosphodiester bond to the 3' side using a distinct mechanism. In this reaction, the vicinal 2'-hydroxyl directly attacks the phosphate group in a manner that is reminiscent of the pancreatic RNase mechanism. The Flp protein can also be shown to possess a topoisomerase-like activity.

Base Sequence↗
PubMed1998

The 2 micrometer plasmid stability system: analyses of the interactions among plasmid- and host-encoded components.

The stable inheritance of the 2 micrometer plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2 micrometer circle-derived plasmid shows relatively poor stability.

Activating Transcription Factors↗
PubMed1997

Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity.

The Flp site-specific recombinase assembles its active site by recruiting the catalytic tyrosine (Tyr-343) from one Flp monomer into the pro-active site containing a triad of Arg-191, His-305, and Arg-308 from a second monomer. In principle, two active sites may be assembled from a Flp dimer by simultaneous, reciprocal contribution of the shared amino acids by its constituent monomers. In practice, only one of the two active sites is assembled at a time, as would be consistent with a recombination mechanism involving two steps of single-strand exchanges. By using substrates containing strand-specific base bulges, we demonstrate that the relative disposition of their DNA arms can account for this active site exclusion. We also show that the exclusion mechanism operates only at the level of positioning Tyr-343 with respect to the pro-active site, and not at the level of orienting the labile phosphodiester bond within the DNA chain. It is not negative cooperativity of substrate binding but, rather, the substrate-induced negative cooperativity in protein orientation that accomplishes half-of-the-sites activity in the Flp system.

Binding Sites↗
PubMed1997

A tetramer of the Flp recombinase silences the trimers within it during resolution of a Holliday junction substrate.

Recombination catalyzed by the Flp site-specific recombinase involves breakage and joining of four DNA strands between two target substrates. The reaction is carried out in two steps of pairwise strand exchanges by a DNA-protein assembly in which four Flp monomers act cooperatively to execute strand cleavage and joining. Two models for recombination have been proposed. In the trimer model, the two active sites required for each step are assembled from three Flp monomers. In the tetramer (or dimer of asymmetric dimers) model, the two active sites are assembled from four Flp monomers, two monomers each contributing one active site. Experiments in which the two models challenge each other reveal that, within the Flp tetramer arranged on a Holliday junction, the two active sites required for its resolution are derived from all four, rather than three, Flp monomers. Thus, the relative protein subunit configuration of the tetramer silences the trimers within it by excluding them from assembling a functional active site pair.

DNA↗
PubMed1997

Probing Flp: a new approach to analyze the structure of a DNA recognizing protein by combining the genetic algorithm, mutagenesis and non-canonical DNA target sites.

A topological and functional overview of a DNA recognition protein with unknown structure can be achieved by combining three different, but complementary approaches: modeling by the genetic algorithm, functional analysis of mutated variants, and testing the target DNA using non-canonical oligonucleotides. As an example we choose the Flp protein, a site-specific recombinase from Saccharomyces cerevisiae. We derive the topological outline including the DNA binding cleft, examine DNA binding regions by deletional and mutational analysis, and analyze the DNA binding site using 7-deazaadenine, 7-deazaguanine, inosine and 4-O-methylthymine as probes. The combined data offer a comprehensive sketch of a plausible protein architecture for Flp. The structure is detailed enough to verify the prediction accuracy for different peptide regions from pre-existing data and by new experimental design.

Binding Sites↗
PubMed1997

The yeast site-specific recombinase Flp mediates alcoholysis and hydrolysis of the strand cleavage product: mimicking the strand-joining reaction with non-DNA nucleophiles.

The yeast site-specific recombinase Flp is covalently linked to DNA via a 3'-phosphotyrosyl bond during the strand-breakage step of recombination. We show that this phosphotyrosyl diester bond formed between Flp and DNA can serve as the target for alcoholysis or hydrolysis in an Flp-assisted reaction. Flp does not mediate alcoholysis of the labile phosphodiester bond within the DNA chain under our assay conditions. The body of available evidence supports the notion that the alcoholysis/hydrolysis reaction is mechanistically analogous to the strand-joining step of the recombination pathway. The only difference is that the DNA 5'-hydroxyl group that acts as the nucleophile during recombination is substituted by a non-DNA nucleophile. We find that the alcoholysis reaction occurs only within the normal cleavage complex produced by the "shared active site" assembled at the interface of two Flp monomers. Unlike the strand-joining reaction, alcoholysis does not occur on an activated DNA substrate linked at its 3'-phosphate end to a short tyrosyl peptide (not to the full-length Flp), and bound non-covalently by a Flp monomer. However, even in this substrate that mimics the strand-cleaved state, the joining reaction is competitively inhibited by a polyhydric alcohol such as glycerol.

Alcohols↗
PubMed1997

The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus.

The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain. Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.

Cell Compartmentation↗
PubMed1996

Positional information within the Mu transposase tetramer: catalytic contributions of individual monomers.

The strand cleavage and strand transfer reactions of Mu DNA transposition require structural/catalytic contributions from separate polypeptide domains of individual transposase (MuA) monomers within the functional MuA tetramer. Based on catalytic complementation between two inactive MuA variants, we have derived certain rules by which the physical location of a MuA monomer within the transposition complex specifies its role in DNA breakage and transfer. During strand transfer, MuA monomers contributing domain II to the reaction occupy R1 (the subsite proximal to the strand-transferred nucleotide), while those contributing domain IIIalpha occupy R2. The positions of the monomers contributing these two domains appear to be reversed during DNA cleavage.

Bacteriophage mu↗
PubMed1996

Assembly and orientation of Flp recombinase active sites on two-, three- and four-armed DNA substrates: implications for a recombination mechanism.

The normal recombination reaction catalyzed by the Flp (pronounced flip) site-specific recombinase between two full-site DNA substrates requires the action of four recombinase monomers in concert. Each monomer of the recombinase harbors an incomplete active site, and is hence chemically incompetent. In order to organize the strand cleavage pocket, it must accept the catalytic tyrosine (Tyr 343) from a second Flp monomer. We address the issue of the potential modes of assembling the shared active site in substrates containing two, three or four Flp binding arms. In normal full-sites (two Flp binding arms), strand cleavage occurs within a substrate and not across substrates. Flp is able to resolve a Y structure (three Flp binding arms) into linear plus hairpin recombinants. Strand cleavage by Flp in a Y structure and in a Holliday structure (four Flp binding arms) follows the trans rather than the cis mode. Within the context of two normal full-sites, all of the strand cutting patterns are best accommodated by a single-cleavage mode, namely the trans-horizontal mode. The assembly and orientation of a Flp active site is determined by whether two Flp-bound DNA arms have the stacking flexibility to accommodate the relevant protein-protein interactions. These results provide support for a model in which pairs of monomers bound within each of the two DNA partners contribute to the strand cleavage reactions that initiate and terminate a normal recombination event. Thus all four Flp monomers are required to mediate the cleavage/joining events at either end of the strand exchange region.

Base Sequence↗
PubMed1996

Analyses of the first chemical step in Flp site-specific recombination: Synapsis may not be a pre-requisite for strand cleavage.

The site-specific recombination reaction mediated by the Flp recombinase occurs within a protein-DNA complex containing four monomers of Flp and two DNA substrates. The reaction requires that the strand-exchange region (also called the spacer or overlap region) of the recombining partners be perfectly homologous. A single Flp monomer bound to its recognition sequence is sufficient to orient the scissile phosphodiester adjacent to it for the phosphoryl transfer reaction that induces strand breakage. Cleavage is inhibited when two to three spacer positions adjacent to the reactive phosphodiester are non-complementary. This requirement for Watson-Crick base-pairing can be overcome under conditions that promote formation of a Flp-Flp dimer across the spacer sequence. Synapsis between two Flp-occupied DNA substrates does not appear to be a pre-requisite for triggering strand cleavage. The reaction is likely initiated when a functional Flp dimer is established across the spacer within a single recombination target site. In the absence of a compatible partner, the cleavage reaction is quickly reversed by resealing the nick. Therefore accumulation of strand breakages is avoided. Coordinated partner cleavages within a synaptic complex can lead to strand joining across partners, thus leading the system towards recombination. Our results are consistent with the generally accepted view that homology between recombining partners is not tested till after strand cleavage has occurred.

Binding Sites↗
PubMed1995

Enhancer-independent variants of phage Mu transposase: enhancer-specific stimulation of catalytic activity by a partner transposase.

Assembly of the functional tetrameric form of phage Mu transposase (A protein) requires specific interactions between the Mu A monomer and its cognate sequences at the ends of the Mu genome (attL and attR) as well as those internal to it (the enhancer element). We describe here deletion variants of Mu A that show enhancer-independence in the assembly of the strand cleavage complex. These deletions remove the amino-terminal region of Mu A required for its interactions with the enhancer elements. The basal enhancer-independent activity of the variant proteins can be stimulated by a partner variant harboring an intact enhancer-binding domain. By exploiting the identical att-binding, and nonidentical enhancer-binding specificities of Mu A and D108 A (transposase of the Mu related phage D108), we show that the stimulation of activity is enhancer-specific. Taken together, these results suggest that the domain of Mu A that includes the enhancer-interacting region may exert negative as well as positive modulatory effects on the strand cleavage reaction. We discuss the implications of these results in the framework of a recent model for the assembly of shared active sites within the Mu A tetramer.

Attachment Sites, Microbiological↗
PubMed1995

Functional roles of individual recombinase monomers in strand breakage and strand union during site-specific DNA recombination.

The site-specific recombinase Flp from Saccharomyces cerevisiae accomplishes recombination between two target DNA sites by executing a pair of strand exchanges at either end of the strand exchange region. One round of recombination requires the cooperative action of four recombinase monomers. We demonstrate here that, in the presence of the appropriate nucleophiles, a single Flp monomer associated with its binding element can mediate strand cleavage and strand joining at the exchange site phosphate adjacent to it. Our results support a model of recombination in which pairs of Flp monomers reverse catalytic roles to mediate the first and second sets of strand breakage/union reactions. They disfavor a model that involves a relay of recombinase monomers between binding elements to assemble separate active sites for strand cleavage and strand joining. Our data are consistent with the breakage and joining reactions being carried out by a single composite active site in which some residues contribute to both reactions while others contribute to one of the two reactions.

Binding Sites↗
PubMed1995

Junction mobility and resolution of Holliday structures by Flp site-specific recombinase. Testing partner compatibility during recombination.

Absolute homology between partner substrates within the strand exchange region (spacer) is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Recent experiments suggest that 3-base pair homology adjacent to the points of exchange at each end of the spacer is utilized in a base complementarity-dependent strand joining reaction. Homology of the central 2 base pairs of the spacer is also critical, but how homology is tested at these two positions is unknown. We have addressed the role of homology-dependent branch migration in Flp recombination by assaying strand cleavage and resolution in a set of synthetic Holliday junctions in which the branch point is freely or partially mobile through the spacer, or is immobilized at each position within the spacer or immediately flanking it. A strong bias in the direction of Holliday resolution is observed only when the branch point is located just outside the spacer (at the junction of the Flp binding element and the spacer). A significantly smaller bias is noticed when the branch point is frozen immediately adjacent to this position within the spacer. Resolution in these cases is most often mediated by exchange of the scissile phosphodiesters at the branch point or proximal to it, and rarely by exchange of the scissile phosphodiesters distal to it. In light of these and previous results, we discuss possible checkpoints for testing partner compatibility during Flp recombination.

Base Sequence↗
PubMed1995

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.

Base Sequence↗