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M Jayaram

Publications and source records attributed to M Jayaram.

At least 55 records · Page 3Linked to original sources
PubMed1995

A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions.

The functional configuration of Mu transposase (A protein) is its tetrameric form. We present here a model for the organization of a functional Mu A tetramer. Within the tetramer, assembly of each of the two active sites for Mu end cleavage requires amino acid contributions from the central and C-terminal domains (domains II and III respectively) of at least two Mu A monomers in a trans configuration. The Mu enhancer is likely to function in this assembly process by specifying the two monomers that provide their C-terminal domains for strand cleavage. The Mu B protein is not required in this step. Each of the two active sites for the strand transfer reaction is also organized by domain sharing (but in the reverse mode) between Mu A monomers; i.e. a donor of domain II (also the recipient of domain III) during cleavage is a recipient of domain II (and the donor of domain III) during strand transfer. The function of the Mu B protein (which is required at the strand transfer step) and that of the enhancer element may be analogous in that their interactions with Mu A (domain III and domain I alpha respectively) promote conformations of Mu A conducive to strand cleavage or strand transfer.

Bacteriophage mu↗
PubMed1995

Role of partner homology in DNA recombination. Complementary base pairing orients the 5'-hydroxyl for strand joining during Flp site-specific recombination.

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.

Base Composition↗
PubMed1995

Step-arrest mutants of phage Mu transposase. Implications in DNA-protein assembly, Mu end cleavage, and strand transfer.

We describe the isolation and characterization of Mu A variants arrested at specific steps of transposition. Mutations at 13 residues within the Mu A protein were analyzed for precise excision of Mu DNA in vivo. A subset of the defective variants (altered at Asp269, Asp294, Gly348, and Glu392) were tested in specific steps of transposition in vitro. It is possible that at least some residues of the Asp269-Asp294-Glu392 triad may have functional similarities to those of the conserved Asp-Asp-Glu motif found in several transposases and retroviral integrases. Mu A(D269V) is defective in high-order DNA-protein assembly, Mu end cleavage, and strand transfer. The assembly defect, but not the catalytic defect, can be overcome by precleavage of Mu ends. Mu A(E392A) can assemble the synaptic complex, but cannot cleave Mu ends. A mutation of Gly348 to aspartic acid within Mu A permits the uncoupling of cleavage and strand transfer activities. This mutant is completely defective in synaptic assembly and Mu end cleavage in presence of Mg2+. The assembly defect is alleviated by replacing Mg2+ with Ca2+. Some Mu end cleavage is observed with this mutant in the presence of Mn2+. When presented with precleaved Mu ends, Mu A(G348D) exhibits efficient strand transfer activity.

Amino Acid Sequence↗
PubMed1994

Directed protein replacement in recombination full sites reveals trans-horizontal DNA cleavage by Flp recombinase.

One round of site-specific recombination between two DNA partners mediated by the Flp recombinase requires the breakage and reformation of four phosphodiester bonds. The reaction is accomplished by the combined action of four Flp monomers. Within the recombination complex, what is the relative disposition of a Flp monomer with respect to the target diester that it cleaves? To address this question, we have devised a strategy for the targeted orientation of Flp monomers within full-site recombination substrates. Our experimental design is not dependent on 'altered binding specificity' of the recombinase. Analysis of the pattern of DNA cleavage by this method reveals no evidence for DNA cleavage in cis. A Flp monomer bound to its recognition element within the full site does not cleave the scissile phosphodiester bond adjacent to it. Our results are most consistent with 'trans-horizontal cleavage'. Cleavage by Flp occurs at the scissile phosphodiester distal to it, but within the same full site. The general experimental design employed here will be of widespread utility in mechanistic analyses of nucleic acid transactions involving multimeric DNA-protein assemblies.

Base Sequence↗
PubMed1994

DNA-protein cooperativity in the assembly and stabilization of mu strand transfer complex. Relevance of DNA phasing and att site cleavage.

The requirements for negatively supercoiled DNA substrates, the cis-acting transposition enhancer and the Escherichia coli HU protein during the phage Mu transposition reaction are relaxed under DMSO-assay conditions. We have used these modified assay conditions to extend studies on the transposition pathway. We show here that linear DNA fragments containing the right end of Mu (attR) and Mu A protein mutually promote the assembly of "high-order" complexes held together by non-covalent protein-DNA and protein-protein interactions. A large subset of these complexes is competent in mediating strand transfer. DNA fragments containing the left end of Mu (attL) as well as non-Mu DNA can be used as targets during strand transfer. The R1 and R2 subsites within attR are required, but R3 is dispensable, in the protein-DNA oligomerization steps as well as in the strand transfer reaction. Proper phasing and spacing between R1 and R2 are central to the reaction. A single base-pair change in the terminal nucleotide that renders attR non-cleavable prevents the assembly of stable high-order complexes, showing that strand cleavage and stabilization of high-order complexes are tightly coupled events. Conversely, pre-cleavage at the attL site allows it to function in the assembly process, albeit at a much lower efficiency than attR. In the presence of HU, the reactivity of pre-cleaved attL is enhanced significantly.

Attachment Sites, Microbiological↗
PubMed1994

Generality of the shared active site among yeast family site-specific recombinases. The R site-specific recombinase follows the Flp paradigm [corrected].

Mutations of the invariant Int family tetrad residues, the RHR triad, and the active site tyrosine, within the Zygosaccharomyces rouxii site-specific recombinase R cause the same "step-arrest" phenotypes as they do in the Flp recombinase of Saccharomyces cerevisiae. In "half-site" recombinations, the R recombinase exhibits catalytic complementation between an RHR triad mutant and an active site tyrosine mutant. Strand cutting by R follows the "trans" DNA cleavage rule. These results are best explained by the assembly of a functional active site from partial active sites harbored by the ARg monomers. Complementation tests using single and double step-arrest ARg mutants verify critical predictions of the "shared active site" model. A wild type monomer paired with an RHR triad-Tyr358 double mutant is a catalytically inactive combination. Pairwise combinations of a single or a double RHR mutant with R(Y358F) yield comparable levels of catalytic complementation. These results strongly imply conservation of the mechanism of active site assembly and the mode of substrate cleavage within the yeast family site-specific recombinases, and perhaps within the larger Int family recombinases [corrected].

Arginine↗
PubMed1994

Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms.

The basic chemistry involved in DNA recombination, RNA splicing and DNA transposition is a phosphoryl transfer reaction. This review is an attempt to provoke a unified thinking on the reaction mechanisms in these nucleic acid transactions. Some of the recent results with the Flp site-specific recombinase that reveal how the chemical reactivity for recombination is derived from cooperative protein-subunit interactions on the DNA substrate are discussed. At least some of the features of Flp reaction are likely to have global implications in other DNA and RNA strand-transfer systems.

Bacterial Outer Membrane Proteins↗
PubMed1994

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.

Base Sequence↗
PubMed1994

Role of tyrosine phosphorylation-dephosphorylation in copy number control of the yeast plasmid 2 micron circle.

A key feature of the copy control in the 2 micron circle plasmid of Saccharomyces cerevisiae is its ability to amplify when the copy number drops below the steady state value. The Flp protein encoded by the plasmid is an essential component of the amplification mechanism. A central regulatory event in amplification involves the phosphorylation/dephosphorylation of Tyr-343 of Flp. Tyrosine phosphorylation is achieved by a transesterification mechanism involving a specific phosphodiester within the 2 micron circle. The dephosphorylation is also a transesterification reaction that uses a specific 5'-OH (generated during tyrosine phosphorylation) as the phosphoryl acceptor. A sum of four phosphorylation/dephosphorylation reactions, coordinated in sets of two, is thought to invert the relative directions of a pair of replication forks. This allows more than one copy of the plasmid to be made from a single replication initiation event. In this paper we discuss the structural features of the Flp active site that control and coordinate the transesterification reactions required for amplification.

Base Sequence↗
PubMed1993

Chitin synthase-encoding gene(s) of the Zygomycete fungus Phycomyces blakesleeanus.

Using polymerase chain reaction (PCR) primers to two highly conserved sequences within fungal chitin synthase (CHS)-encoding genes, an approximately 750-bp DNA fragment was amplified from genomic DNA of Phycomyces blakesleeanus. The amino acid sequence deduced from the nucleotide sequence of this fragment best matches the motifs found in class-II CHS. The fragment includes an approximately 160-bp region that likely is an intron. Southern hybridization of restriction enzyme-digested genomic DNA, using the PCR-amplified DNA as the probe, suggests that P. blakesleeanus contains additional CHS-encoding genes (CHS). To our knowledge, this is the first report on the detection of a CHS gene in a Zygomycete fungus. These studies represent a major step toward exploring the molecular mechanisms of CHS regulation in Phycomyces. The prospects are exciting, since CHS is implicated to play a central role in the sensory responses of P. blakesleeanus involving growth modulations.

Amino Acid Sequence↗
PubMed1993

Mechanism of site-specific recombination. Logic of assembling recombinase catalytic site from fractional active sites.

The active nucleophilic species in the strand cleavage and strand exchange steps of site-specific recombination by the Flp protein are the active site tyrosine (Tyr-343) of Flp and the 5'-hydroxyl of Flp-nicked DNA, respectively. The target phosphodiester, activated by Flp, can be cleaved by an exogenous nucleophile derived, for example, from H2O2. Flp variants that are defective in the phosphate activation step and cannot sustain Tyr-343-mediated cleavage also fail to elicit H2O2-mediated cleavage. An Flp mutant lacking Tyr-343, (Flp(Y343F)), can carry out both the strand cleavage and strand exchange reactions in the presence of a age and strand exchange reactions in the presence of a tyrosine analog. These results are consistent with a cis-activation/trans-nucleophilic attack paradigm for strand breakage and strand union. The proposed model conceptually unifies the chemistry and enzymology of the two partial reactions of recombination. The mechanism of Flp action has strong implications for phosphoryl transfer reactions in other site-specific DNA recombination systems and in RNA splicing.

Base Sequence↗
PubMed1993

Tests for the fractional active-site model in Flp site-specific recombination. Assembly of a functional recombination complex in half-site and full-site strand transfer.

The Arg191-His305-Arg308 (the RHR triad) and Tyr343 of Flp site-specific recombinase correspond to the invariant tetrad residues of the integrase family of proteins. Flp mutants altered at these positions are blocked at the strand cleavage or the strand exchange step of recombination. Hybrid half-site-recombinase complexes formed by step-arrest mutants of Flp have revealed that an Flp monomer occupying a half-site does not cleave that half-site but rather cleaves a half-site occupied by a second Flp monomer. This trans-DNA cleavage is neatly accommodated by a model in which an Flp active site is assembled by contribution of amino acid residues from at least two protein monomers. Using a combination of wild type Flp, single, double, and triple step-arrest Flp mutants, critical predictions of the fractional active-site model have been verified. First, a wild type monomer paired with an RHR triad-Tyr343 double mutant is a catalytically inactive combination. Second, each pairwise combination of a single, double, or triple RHR mutant with Flp (Y343F) yields approximately equivalent levels of catalytic complementation. Half-site to half-site and half-site to full-site crosses suggest that execution of a strand transfer event within a half-site and between a half-site and a full site requires dimeric and tetrameric Flp configurations, respectively.

Amino Acid Sequence↗
PubMed1993

Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase.

Hydrogen peroxide is capable of effecting the cleavage of a specific phosphodiester bond in DNA, when used in concert with the recombinase enzyme Flp from Saccharomyces cerevisiae. This cleavage is not caused by oxidative damage of the DNA backbone but instead is the result of nucleophilic attack by peroxide. A single phosphorus-oxygen bond is broken in the reaction. Cleavage of DNA by peroxide also occurs with an inactive mutant of Flp in which the active site nucleophile tyrosine has been replaced by phenylalanine. Besides providing information on the mechanism of strand cleavage by Flp, these results may contribute to the development of new synthetic DNA cleavage reagents that act by hydrolytic and not radical chemistry.

Base Sequence↗
PubMed1993

DNA splicing by an active site mutant of Flp recombinase. Possible catalytic cooperativity between the inactive protein and its DNA substrate.

Each strand transfer catalyzed by the Flp recombinase is the composite of two transesterification reactions. The active nucleophilic species in the two reactions are the catalytic site tyrosine (Tyr-343) of Flp and the 5'-hydroxyl from the Flp-nicked DNA substrate, respectively. A "half recombination site" is capable of undergoing this pair of transesterifications in the presence of Flp. When the substrate is a half-site containing a chiral phosphorothioate at the exchange point, the Flp reaction yields a product in which the phosphate chirality is retained. A mutant of Flp that lacks the active site tyrosine, Flp(Y343F), is incapable of mediating strand transfer in a full-recombination site but can execute strand transfer in a half-site. The efficiency of this reaction is about 2% of that of the wild type reaction. The activity of Flp(Y343F) is critically dependent on the length of the half-site spacer. Furthermore, in this reaction, the strand cleavage and strand exchange steps cannot be uncoupled. These results strongly suggest a direct attack by the 5'-hydroxyl of the half-site spacer on the phosphodiester at the normal strand transfer point.

Base Sequence↗
PubMed1992

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They 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 as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).

DNA Mutational Analysis↗
PubMed1992

Bending-incompetent variants of Flp recombinase mediate strand transfer in half-site recombinations: role of DNA bending in recombination.

One key feature of the interaction of Flp recombinase with its target site (FRT) is the large bend introduced in the substrate as a result of protein binding. The extent of bending was found to depend on the phasing and spacing of the Flp monomers occupying the two Flp-binding elements (FBE) bordering the strand-exchange region (spacer) of the substrate. The relative mobilities of the Flp complexes formed by the two permuted substrate fragments, containing the FRT site near the end or in the middle, corresponded to a DNA bend of approx. 140 degrees when each of the two FBEs flanking the spacer was occupied by a protein monomer. The estimated bend angle was the same when the reference DNA fragment with the FRT site at the end was substituted by one with the site in the middle, but containing a 4-bp insertion within the spacer. We used a combination of wild-type Flp and Flp variants that were competent or incompetent in DNA bending, together with full, or half FRT sites, to ask whether bending is a conformational requirement for catalysis, namely cleavage and exchange of strands. We obtained the following results: in full-site (FRT) vs. full-site recombinations or in full-site vs. half-site (half FRT) recombinations, there was a large difference in the reactivity between Flp and a bending-incompetent Flp variant. This difference virtually disappeared when reactions were done with half-FRT sites. We conclude that bending is not a prerequisite for catalysis, but represents the manner in which the substrate accommodates the Flp protomer-protomer interactions that are pertinent to catalysis.

Base Sequence↗