Homologous recombination and RecA protein: towards a new generation of tools for genome manipulations

doi:10.1016/j.tibtech.2004.12.005

Trends in Biotechnology

Volume 23, Issue 2, February 2005, Pages 97-102

https://doi.org/10.1016/j.tibtech.2004.12.005 Get rights and content

Homologous recombination (HR) is one of the central processes of DNA metabolism, combining roles in both cell housekeeping and the evolution of genomes. In eukaryotes, HR underlies meiosis and ensures genome stability. The complete sequencing of numerous bacterial genomes has shown that HR has a substantial role in the evolution of microorganisms, especially pathogens. HR systems from different species and their isolated components are finding an expanding field of applications in modern genetic engineering and bio- and nanotechnologies. Recently, much progress has been made in our understanding of HR mechanisms in eukaryotes and the practical applications of HR systems.

Section snippets

HR: where it works, housekeeping and global evolution

Although by definition HR is a pathway for gene shuffling and exchange between cells, data indicating that the HR system has an additional role in DNA repair began to accumulate almost immediately after the identification of genes involved in recombination (reviewed in Ref. [1]). These data eventually led to the concept of recombination-dependent repair (RDR). The main distinguishing characteristic of RDR is that undamaged homologous DNA is used as a template for repair. This feature

RecA protein: recombinational filaments and DNA strand exchange

RecA protein from E. coli has functional and structural analogs in all kingdoms of life 2, 27. These proteins share a universal principle for structural organization of the main recombination intermediate – that is, they all form a spiral nucleoprotein or recombinational filament called a ‘presynaptic complex’ [28]. They also show a common preference for binding to specific DNA sequences (Box 1). These findings demonstrate that the fundamental mechanisms of HR are common to all forms of life.

Applications of RecA protein and endogenous recombinational systems

The main cellular function of RecA protein is the recognition of homology between DNA molecules. In contrast to traditional approaches that rely on the spontaneous annealing of two ssDNA molecules, RecA promotes homologous pairing between ssDNA and dsDNA substrates. This form of homology recognition can provide a substantial advantage over conventional hybridization methods by avoiding potential damage during DNA denaturation.

This approach also has a more fundamental difference. For

RecA-assisted directed DNA cleavage and ligation

The method for specific DNA cleavage using RecA was developed in two laboratories (reviewed recently in Refs 36, 37) and now is known by two names: RARE and RecA-AC. According to the suggested procedure, the presynaptic complex of RecA protein and its corresponding oligonucleotide is used to find the homologous target on long dsDNA. In this approach, this target must contain a site of methylation or restriction, and the cognate methylase must possess some activity under conditions of stable

RecA-assisted hybridization and selective cloning

RecA complexes have been also used for the direct physical trapping of homologous DNA from mixtures of dsDNA fragments, for which a biotinylated ssDNA probe is used. After the RecA-promoted formation of specific complexes with the DNA target, the latter can be isolated on streptavidin-conjugated paramagnetic beads. Kits for enriching cDNA libraries through the use of RecA-coated biotinylated DNA probes are commercially available (see ClonCapture™ cDNA Selection, //www.bdbiosciences.com/clontech/archive/OCT99UPD/pdf/ClonCapApNote.pdf

In vivo uses of RecA–DNA complexes

An appealing idea is to use RecA protein complexes for gene targeting in vivo. Such attempts have been described recently by Maga et al. [49], who found that the injection of RecA-coated DNA into porcine and caprine zygotes substantially improved both transgene integration efficiency and embryo survival. They proposed that this effect results not from the catalytic activity of the introduced complexes, but from the protection of the incoming DNA from degradation within the nucleus and the

Concluding remarks

Recent studies have shed light on the place of HR among other basic genetic systems and its mechanisms in different species, particularly in eukaryotes. HR is emerging as a process that has extremely widespread implications in areas as diverse as chromosome segregation in higher eukaryotes and the adaptational behavior of populations of bacteria. Tremendous progress has been made in elucidating several pathways of HR in different species. Although at present our knowledge of the mechanism of HR

Acknowledgements

Space limitations have restricted us to the citation of recent reviews instead of primary publications in places, and we apologize for omitting references to many original contributions. We are grateful to Masayuki Takahashi for sending us his review before publication. A.A.V. is supported by a grant from the Russian Foundation for Basic Research (04-04-48869-a).

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Cited by (14)

Reversibility, equilibration, and fidelity of strand exchange reaction between short oligonucleotides promoted by RecA protein from escherichia coli and Human Rad51 and Dmc1 proteins
2009, Journal of Biological Chemistry
We demonstrate the reversibility of RecA-promoted strand exchange reaction between short oligonucleotides in the presence of adenosine 5′-O-(thiotriphosphate). The reverse reaction proceeds without the dissociation of RecA from DNA. The reaction reaches equilibrium and its yield depends on the homology between the reaction substrates. We estimate the tolerance of the RecA-promoted strand exchange to individual base substitutions for a comprehensive set of possible base combinations in a selected position along oligonucleotide substrates for strand exchange and find, in agreement with previously reported estimations, that this tolerance is higher than in the case of free DNA. It is demonstrated that the short oligonucleotide-based approach can be applied to the human recombinases Rad51 and Dmc1 when strand exchange is performed in the presence of calcium ions and ATP. Remarkably, despite the commonly held belief that the eukaryotic recombinases have an inherently lower strand exchange activity, in our system their efficiencies in strand exchange are comparable with that of RecA. Under our experimental conditions, the human recombinases exhibit a significantly higher tolerance to interruptions of homology due to point base substitutions than RecA. Finding conditions where a chemical reaction is reversible and reaches equilibrium is critically important for its thermodynamically correct description. We believe that the experimental system described here will substantially facilitate further studies on different aspects of the mechanisms of homologous recombination.
Purification and characterization of the Thermus thermophilus HB8 RecX protein
2007, Protein Expression and Purification
The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the α-helical content of ttRecX is 54% and that it is stable up to 80 °C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.
Calorimetric Analysis of Binding of two Consecutive DNA Strands to RecA Protein Illuminates Mechanism for Recognition Of Homology
2007, Journal of Molecular Biology
RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (ΔH) with temperature (heat capacity change, ΔC_p). In the presence of the ATP analogue ATPγS, the ΔH for the binding of the first DNA strand depends upon temperature (large ΔC_p) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced ΔC_p, indicating the absence of further reorganisation of the RecA–DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the ΔH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the ΔH is larger than that for non-complementary DNA strand, but less than the ΔH of the annealing of the complementary DNA without RecA. This small ΔH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The ΔH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.
A novel single step double positive double negative selection strategy for β-globin gene replacement
2006, Biochemical and Biophysical Research Communications
Citation Excerpt :
This rate is estimated to be only 10−5 to 10−7[18]. Some approaches have been employed to improve the efficiency of HR such as, transient overproduction of an active recombinase like RecA or RAD51 which could increase the HR rate [33,34]. Recently, sequence-specific zinc finger nucleases have been used to induce double strand breaks in desired sites [35].
β-Thalassemias are a heterogeneous group of autosomal recessive disorders, characterized by reduced or absence of the β-globin chain production by the affected alleles. Transplantation of genetically corrected autologous hematopoietic stem cell (HSC) is an attractive approach for treatment of these disorders. Gene targeting (homologous recombination) has many desirable features for gene therapy due to its ability to target the mutant genes and restore their normal expression. In the present study, a specific gene construct for β-globin gene replacement was constructed consisting of: two homologous stems including, upstream and downstream regions of β-globin gene, β-globin gene lying between hygromycin and neomycin resistant genes as positive selection markers and thymidine kinase expression cassettes at both termini as negative selection marker. All segments were subcloned into pBGGT vector. The final plasmid was checked by sequencing and named as pFBGGT. Mammalian cell line COS-7 was transfected with linear plasmid by lipofection followed by positive and negative selection. DNA of the selected cells was analyzed by PCR and sequencing to confirm the occurrence of homologous recombination. In this novel strategy gene replacement was achieved in one step and by a single construct.
"cre/loxP plus BAC": A strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens
2016, Scientific Reports
In vitro gene manipulation of spinal muscular atrophy fibroblast cell line using gene-targeting fragment for restoration of SMN protein expression
2016, Gene Therapy

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Trends in Biotechnology

Homologous recombination and RecA protein: towards a new generation of tools for genome manipulations

Section snippets

HR: where it works, housekeeping and global evolution

RecA protein: recombinational filaments and DNA strand exchange

Applications of RecA protein and endogenous recombinational systems

RecA-assisted directed DNA cleavage and ligation

RecA-assisted hybridization and selective cloning

In vivo uses of RecA–DNA complexes

Concluding remarks

Acknowledgements

Trends Biochem. Sci.

Mutat. Res.

Curr. Opin. Microbiol.

Theor. Popul. Biol.

Curr. Opin. Microbiol.

Res. Microbiol.

J. Biol. Chem.

Semin. Immunol.

Trends Biochem. Sci.

FEBS Lett.

Trends Biochem. Sci.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

Curr. Opin. Biotechnol.

Mol. Cell. Neurosci.

Genomics

Mutat. Res.

Exp. Cell Res.

Curr. Opin. Genet. Dev.

Curr. Opin. Plant Biol.

Trends Biochem. Sci.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Mol. Biol.

J. Biol. Chem.

FEBS Lett.

Trends Biochem. Sci.

Historical overview: searching for replication help in all of the rec places

Proc. Natl. Acad. Sci. U. S. A.

Role of recombination enzymes in mammalian cell survival

Proc. Natl. Acad. Sci. U. S. A.

Homologous DNA recombination in vertebrate cells

Proc. Natl. Acad. Sci. U. S. A.

Molecular views of recombination proteins and their control

Nat. Rev. Mol. Cell Biol.

Post-replication repair in DT40 cells: translesion polymerases versus recombinases

BioEssays

Repair of radiation damage to DNA

Br. J. Cancer

Recombination within natural populations of pathogenic bacteria: short-term empirical estimates and long-term phylogenetic consequences

Proc. Natl. Acad. Sci. U. S. A.

Lateral gene transfer: when will adolescence end?

Mol. Microbiol.

Horizontal gene transfer: a critical view

Proc. Natl. Acad. Sci. U. S. A.

On the evolution of cells

Proc. Natl. Acad. Sci. U. S. A.

A new biology for a new century

Microbiol. Mol. Biol. Rev.