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Structural studies of the FLP recombinase of saccharomyces cerevisiae complexed with DNA

Karen Hildegard Luetke

Structural studies of the FLP recombinase of saccharomyces cerevisiae complexed with DNA

implications for the mechanism of site-specific recombination.

by Karen Hildegard Luetke

  • 231 Want to read
  • 15 Currently reading

Published .
Written in English


The Physical Object
Pagination233 leaves.
Number of Pages233
ID Numbers
Open LibraryOL19517662M
ISBN 100612352331

This chapter starts with Allan Campbell’s insightful proposal for the pathway by which the chromosome of bacteriophage λ is integrated into and excised from the chromosome of its Escherichia coli host. The chapter discusses different levels of λ Int family complexity. There are presently four classes of integron integrases, IntI1 to -4, sharing approximately 50% identity. The Å structure of Cre recombinase bound to an immobile HJ and the Å structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold‐symmetric DNA intermediate that shares features with both the stacked‐X and the square conformations of the HJ that exist in the unbound state.


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Structural studies of the FLP recombinase of saccharomyces cerevisiae complexed with DNA by Karen Hildegard Luetke Download PDF EPUB FB2

The present study focuses on the mechanism of recombination by the Flp recombinase coded for by the Saccharomyces cerevisiae plasmid two micron circle.

The biological function of Flp is to trigger a replication-coupled DNA amplification reaction, which corrects any decrease in plasmid copy number resulting from rare missegregation events (2, 4).Cited by: and the eukaryotic FLP recombinase of the yeast Saccharomy-ces cerevisiae (9, 51).

Common to all these systems are two different or identical recognition sites that serve as the DNA substrate for the recombinase. In recent years, recombinases have become important tools to manipulate genetically prokaryotes as well as eukaryotes.

InCited by:   The FLP/ FRT recombination system is encoded by the 2μm ( kb) plasmid that is present in most isolates of S. cerevisiae. Due to its stable partitioning and amplification system, this plasmid is available with a copy number of 60 to copies per cell (16, 63).Cited Structural studies of the FLP recombinase of saccharomyces cerevisiae complexed with DNA book   Part of the plasmid amplification system, which corrects any decrease in copy number caused by a rare missegregation event.

Catalyzes the recombination between the large inverted repetitions of the 2-micron plasmid during plasmid replication. This recombination event changes the direction of one of the two replication forks in the bidirectionally replicating molecule, effectively. Flippase (Flp), another site-specific recombinase for manipulating DNA, was developed from Saccharomyces cerevisiae (Chen and Rice, ).

The Flp recombinase. The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid to that is conserved among several FLP-like proteins from different yeasts.

Gronostajski RM, Sadowski PD. The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

Mol Cell Biol. Nov; 5 (11)– [PMC free article] Guo F, Gopaul DN, van Duyne GD. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.

Nature. We propose that, regardless of cis or trans cleavage, the active species is a recombinase dimer constituted by monomers bound to the left and right binding arms of a DNA substrate.

Solution studies with Flp (Lee et al., ; Voziyanov et al., ) and the protein contacts observed in the structure of the DNA‐bound Cre tetramer (Guo et al. Members of the integrase (Int) 1 family of site-specific recombinases (λ-integrase, P1 Cre recombinase,Escherichia coli XerC/XerD recombinase, Saccharomyces cerevisiae Flp, and Zygosaccharomyces rouxii R recombinases among several others) all carry out conservative site-specific recombination using a basic type IB topoisomerase reaction scheme (1, 2).

The DNA recombination sequences where recombinase binding and strand exchange take place are referred to as the core recombination sites. These sites are composed of two recombinase binding elements (RBEs) arranged as inverted repeats surrounding a central strand exchange or crossover region.

A parallel alignment of sites is defined as one in which the DNA helical axes of the crossover. Nucleotide heterogeneity at position in Saccharomyces cerevisiae 25S rRNA. LC-MS and MS 2 of the RNase T1 digest of U/C-5D-labelled and reverse-phase LC-purified 25S rRNA ( fmol). (A) Extracted ion monitoring of fragments containing a nt at position shows that nt is a mixture containing 9% U, 74% Um, 4% Ψ and 13% Ψm (Table 2).The extracted ion masses have m/z values.

DNA recombinase enzymes such as Cre or FLP recombinases detect specific flanking sequences that, depending on their orientation, allow for excision or rotation of the flanked DNA segment This mechanism can be used to activate/deactivate a gene depending on the presence of the matching recombining enzyme, which allows for recombinase-dependent viral vector delivery of opsins.

Saccharomyces cerevisiaeMus81 is a structure-selective endonuclease which constitutes an alternative pathway in parallel with the helicase-topoisomerase Sgs1-Top3-Rmi1 complex to resolve a number of DNA intermediates during DNA replication, repair, and homologous recombination.

The Flp-nick system is a simple in vivo system developed for studying the cellular responses to a protein-bound nick at a single genomic site in the budding yeast Saccharomyces cerevisiae. The Flp. The Flp site-specific recombinase from Saccharomyces cerevisiae induces DNA bending upon interaction with the Flp recognition target (FRT) site.

The minimal FRT site is comprised of two inverted binding elements which flank a central core region. Binding of a single monomer of Flp to DNA induces a DNA bend of 60 degrees. A hindrance to the study of structure and function of DNA elements is that sites of interest always lie within the context of other DNA sequences.

Formation of Extrachromosomal DNA Rings in Saccharomyces cerevisiae Using Site-Specific Recombination. Authors; K. and Lindquist, S. () The FLP recombinase of yeast catalyzes site. Flp is a tyrosine recombinase from Saccharomyces cerevisiae that specifically recognizes a pair of bp inverted repeats and catalyzes recombination of the intervening sequence.

The recombination reaction involves two rounds of DNA cleavage, strand exchange and religation (7). The Flp protein from Saccharomyces cerevisiae is one of the site-specific tyrosine family recombinases that are used widely in genomic engineering.

As a first step towards mediating directed DNA rearrangements at non-native Flp recombination targets (mFRTs), we have evolved three separate groups of Flp variants that preferentially act on mFRTs containing substitutions at the first.

The site‐specific recombinase FLP from Saccharomyces cerevisiae is used as a tool for genetic manipulations in a wide variety of organisms from bacteria to mammalian cells (for a review see 16). We have genetically engineered the FLP gene for expression in C.

albicans and used it as a reporter of C. albicans virulence gene expression (29). The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange.

In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G2/M phase, which has been widely assumed to be DNA replication.

To determine whether DNA replication through a silent mating-type locus, HMR a, is required for. The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer.

Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has.

Flp, encoded by the 2 μm plasmid of the yeast Saccharomyces cerevisiae, is a member of the λ integrase or tyrosine-based family of site-specific DNA enzymes use the same basic chemistry under different circumstances to accomplish a variety of tasks, including inserting bacteriophage DNA into host genomes, altering patterns of gene expression, mobilizing certain mobile.

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT).

FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl Saccharomyces cerevisiae, Flp recombinase is a prototypical tyrosine recombinase that catalyzes intramolecular recombi-nation between two inverted bp repeats within the yeast 2-mM DNA plasmid (Broach and Hicks, ; Broach etal.,;HartleyandDonelson,;Fig.3).Theminimal bp Flp recognition target (FRT) site consists of two bp.

Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: /ch During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites.

The bacteriophage recombinase Cre catalyses site-specific recombination between two base-pair loxP. The Int superfamily of recombinases encompasses over members (7,8), including the Flp recombinase of Saccharomyces cerevisiae and the Cre recombinase from bacteriophage P1.

As a genetic-engineering tool, the Cre system has had profound impact on our basic understanding of biological systems and the development of useful animal models of. DNA recombinase FLP gene exists on the 2 micro plasmid of Saccharomyces cerevisiae. Recombinase FLP could recognize an FRT site composed of.

Among more than a hundred known members of this family, extensive biochemical and structural information is available for a small subset—Int proteins from phages λ and HP1, XerC/XerD from Escherichia coli, Cre from phage P1 and Flp from the Saccharomyces cerevisiae plasmid 2 micron circle.

Tyrosine recombinases, whose name derives from the. The first part of this chapter is devoted to the 2µm circle partitioning system, a critical component of the plasmid's strategy for stable maintenance in yeast populations.

The second part deals with plasmid copy number control, special attention being paid to the Flp recombination system that is believed to trigger a DNA amplification process. Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges.

The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr Arg, His, and Arg appear to facilitate the cleavage and exchange steps of recombination.

The recently determined structure of a Flp-DNA complex confirms that its catalytic domain closely resembles that of other family members, although the active site itself is assembled differently.

The reaction mechanism as drawn is the distillation of years of work by many laboratories. In addition to the structure of the Flp-DNA complex, which is the focus of this chapter, crystal structures.

In this study, we generated AxCAFLP, a rAd expressing FLP recombinase derived from Saccharomyces cerevisiae and carried out quantitative comparisons with Cre-expressing rAd in both in vitro and in cultured cells to provide another efficient gene regulation system in mammalian cells.

Flp Recombinase System. In addition to the Cre-loxP system, two other recombinase systems have been used successfully in vivo: the Flp-FRT system from the budding yeast Saccharomyces cerevisiae 34 and α-integrase.

Flp recombinase recognizes a bp consensus sequence known as FRT and induces recombination between two of these sites. The. In this study we focus on understanding substrate recogni-tion in a site-specific DNA recombinase.

Flp is a tyrosine recombinase from Saccharomyces cerevisiae that specifically recognizes a pair of bp inverted repeats and catalyzes recombination of the intervening sequence. The recombina-tion reaction involves two rounds of DNA cleavage, strand. Saccharomyces cerevisiae Mus81 is a structure-selective endonuclease which constitutes an alternative pathway in parallel with the helicase-topoisomerase Sgs1-Top3-Rmi1 complex to resolve a number of DNA intermediates during DNA replication, repair, and homologous recombination.

Previously, it was showed that the N-terminal region of Mus81 was required for its in vivo function in a redundant. As a member of the wwPDB, the RCSB PDB curates and annotates PDB data according to agreed upon standards. The RCSB PDB also provides a variety of tools and resources.

Users can perform simple and advanced searches based on annotations relating to sequence, structure and function. These molecules are visualized, downloaded, and analyzed by users who range from students to specialized scientists.

FLP recomblnase Xiao-hong Qian and Michael M. Cox ~ Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin USA The FLP recombinase promotes a site-specific recombination reaction in the 2it plasmid of yeast.

The protein-DNA complex. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, (Makkuni Jayaram, Ian Grainge and Gena Tribble) Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex, (Phoebe A.

Rice) Bacterial Site-Specific DNA. In mice and mammalian cell cultures, the use of site-specific DNA recombinases, such as FLP from Saccharomyces cerevisiae and Cre from bacteriophage P1, to manipulate genomic DNA by DNA rearrangement is a well-established approach.

Both FLP and Cre recombinases can be applied to alter gene structures to either ablate or turn on gene activities.recombinase of bacteriophage P1 and the Flp recombinase of Saccharomyces cerevisiae, follow the type IB topoisomerase chemistry to promote DNA breakage and union [1–3].

During strand cleavage, the active site tyrosine nucleophile becomes covalently linked to the phosphate end of the broken DNA [3,4]. The hydroxyl group.The Flp recombinase maintains the high copy number of Saccharomyces cerevisiae plasmid 2μm by recombining two identical bp minimal frt sites (Fig.

1C). In contrast to the lambda system, both the Cre and Flp proteins are simple recombination systems requiring no accessory proteins or sequences.