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
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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2007, Journal of Molecular BiologyA novel single step double positive double negative selection strategy for β-globin gene replacement
2006, Biochemical and Biophysical Research CommunicationsCitation 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].