Makkuni Jayaram

Step-arrest mutants of FLP recombinase: Implications for the catalytic mechanism of DNA recombination

The site-specific recombinase (FLP) encoded by the yeast plasmid 2 micron circle belongs to the integrase (of phage lambda) family of recombinases. The sparse homology within the members of this family contrasts with the invariance of three residues, His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them. We report here results on substrate recognition and catalysis by FLP proteins altered at these residues. Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination. We provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate.

Properties of REP3: a cis-acting locus required for stable propagation of the Saccharomyces cerevisiae plasmid 2 microns circle.

Stable propagation of the yeast plasmid 2 microns requires an origin of replication, a cis-active locus designated REP3, and two plasmid-encoded proteins which are the products of the REP1 and REP2 genes. The three REP loci appear to constitute a partitioning system, ensuring equal distribution of plasmid molecules to mother and daughter cells after mitosis. We have localized the REP3 site completely within a segment of five-and-one-half direct tandem repeats of a 62-base-pair unit, bordered by HpaI and AvaI restriction sites within the large unique region of the 2 microns genome. In addition, we find that the repeated elements are functionally distinct. Only a subset of the repeats is necessary to promote full partitioning activity. The other repeats appear to promote plasmid transcription. These results are discussed in the context of a model of plasmid copy control involving titration of a plasmid-specific protein by the repeated elements within REP3.

Phycomyces blakesleeanus TRP1 gene: organization and functional complementation in Escherichia coli and Saccharomyces cerevisiae

We have cloned the gene encoding the TRPF and TRPC functions of Phycomyces blakesleeanus by complementation of the corresponding activities of Escherichia coli. TRPF also complemented a trpl mutation in Saccharomyces cerevisiae. As in other filamentous fungi, such as Neurospora and Aspergillus spp., the P. blakesleeanus TRPF and TRPC formed part of a trifunctional polypeptide encoded by a single gene (called TRP1). Transcription of TRP1 in P. blakesleeanus did not appear to be regulated by light or by the nutritional status of the culture. The information on the structure and organization of a P. blakesleeanus gene derived from these studies should be useful in devising molecular genetic strategies to analyze the sensory physiology of this organism.

Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.

TheInt family of site-specific recombinases: Some thoughts on a general reaction mechanism

The FLP recombinase of the yeast 2 micron circle plasmid belongs to theInt family of recombinases. Only three amino acid residues are invariant among members of this family. Functional analyses of FLP protein variants mutated at these three residues suggest their involvement at specific steps of the recombination pathway. We propose that these residues play the same functional role in the mechanism of action of all theInt family recombinases.

Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.

Rapid localization and characterization of random mutations within the 2μ circle site-specific recombinase: A general strategy for analysis of protein function

A method for rapidly localizing and characterizing mutations within the 2 micron circle site-specific recombinase enzyme (FLP) is described. The strategy consists of dividing the gene coding for FLP (FLP) by artificially introduced unique restriction enzyme sites (that do not alter the amino acid sequence of the protein) into segments of 150-200 bp, mutagenizing the engineered gene and selecting mutants in vivo, localizing each mutation to one of the gene segments by an in vivo complementation assay, and characterizing the mutation by sequencing of only this DNA segment. The experimental designs embodied by this method should be of wide application in investigations of protein function in general and of DNA-protein interactions in particular.

Inducible expression of REP1 causes inducible expression of the 2 micron circle stability system

SummaryThe yeast plasmid, 2 micron circle, encodes a stability system consisting of the plasmid replication origin, a cis-active locus designated REP3 and two transactive functions — the products of the REP1 and REP2 genes. We have constructed 2 micron circle derivatives in which the expression of the REP1 gene is placed under the control of the yeast GAL10 promoter. We show that in such plasmids the stability-system is inducible, being turned off by glucose and turned on by galactose. Further, our results unequivocally demonstrate that, of the two potentialin-frame ATG codons at which REP1 translation might initiate (as inferred from the 2 micron circle DNA sequence and from the cap site of the major REP1 transcript), the upstream ATG is dispensable without affecting REP1 function. We also illustrate here a simple and general method for constructing in vivo in yeast 2 micron circle analogs which contain desired alterations within specific regions of the 2 micron circle genome.