Definition of Recombinase

Recombinase is a family of enzymes that catalyze site-specific recombination events within DNA.

Site-specific DNA recombination is a process in which DNA strands are broken into pieces and then recombined in different combinations of alleles (variant forms of genes), having some sequence similarity.

Site-specific recombinases rearrange DNA segments by first identifying and binding to specific or target sites at which they make the break, exchange the two segments, and then rejoin the strands.

In some site-specific recombination systems, recombinase enzymes are enough to carry out the process of recombination, however, in some others, several other accessory proteins are required to support the system.

The recombinase enzymes were first derived from fungi and bacteriophages (also known as phages) where it’s been involved in many cellular processes including replication, pathogenesis, differentiation, and mobile genetic element movement.

In multicellular organisms, the enzymes are used to modify/manipulate genome structure and control gene expression. They do so by activating four functional mechanisms, which include translocation, inversion, deletion/insertion, and cassette exchange.

Some well known and commonly studied examples of site-specific recombinases are:

• Cre recombinase: a tyrosine recombinase enzyme (also known as type I topoisomerase) isolated from P1 bacteriophages. It catalyzes the reaction of site-specific DNA recombination between two loxP sites. It’s also utilized in developing inducible recombinase systems for genome engineering.
• Hin recombinase: is composed of 198 amino acids and belongs to the serine protease family. The recombinase system is originally found in the bacteria Salmonella, where it performs DNA cleavage and recombination.

It inverts 900 base pairs, containing a promoter for downstream flagellar genes, fljA, and fljB, within the bacterial genome that help in escaping host immune responses.

• Tre recombinase: an experimental enzyme formed by modifying Cre recombinase through selective mutations. In lab tests, it has shown the removal of DNA segments, inserted by the human immunodeficiency virus (HIV), from infected cells.
• FLP recombinase: derived from the 2 µ plasmid of baker’s yeast (or Saccharomyces cerevisiae). It performs Flp-FRT recombination, which is used to manipulate DNA sequences in vivo under controlled conditions. Its functions are analogous to the Cre-loxP system (where pLox is a substrate of Cre recombinase).

In this article, we will cover furthermore about the recombinase enzymes, including their types in organisms, functions, and in vitro lab applications.

Recombinase Functions and Importance

Recombinases have multiple functional roles in different organisms. They are involved in genome manipulation, DNA amplification, site-specific and homologous recombination, and escaping host-immune response in bacterial species.

Genome Manipulation

Many recombinases are known from eukaryotic and prokaryotic organisms that, along with other proteins, act on unique asymmetric DNA sequences and cause genetic manipulation. The recombination product depends on the orientation of these sequences.  

The four basic functional mechanisms of recombinases that are involved in genetic manipulation include:

• Excision/insertion: excision is the removal of a segment of double-stranded DNA and insertion is the addition of a nucleotide segment into the genomic DNA of organisms. These events lead to mutagenesis and might cause phenotype or genotype abnormalities in organisms.
• Inversion: a reversal in the original order of the nucleotide sequences of nucleic acids or rearrangements within the chromosomes.
• Translocation: a phenomenon in which one part of a chromosome is transferred to another chromosome.
• Cassette exchange: a process in which a gene cassette flanked by a pair of incompatible target sites is exchanged with another gene cassette flanked by two identical sites.

Site-Specific Recombination

Site-specific recombination is also known as conservative site-specific recombination. It’s a process in which two DNA strands, having a certain degree of sequence homology, exchange segments.

Typically, the two sites between which the recombinase enzymes act and the recombination occur are identical. However, there are also some exceptions to this case, such as attP and attB of λ integrase.

The events that occur during the site-specific recombination process include:

• Site-specific recombinases recognize a short, specific DNA sequence and bind to it.
• The recombinase enzyme cleaves the DNA backbone.
• Segments of DNA strands are exchanged.
• DNA strands are rejoined by the DNA ligase enzyme.

Many genome engineering strategies rely on site-specific recombination, including recombinase-mediated cassette exchange (RMCE). It’s an efficient method for the targeted integration of DNA transcription units into specific genomic loci.

Applications of Recombinase

Site-specific recombinases have several applications in molecular biology and biochemistry labs to perform in vitro assays. It includes genetic manipulation or modification with specificity, cloning, recombinase polymerase amplification using PCR, site-directed fluorescence labeling, developing transgenic organisms, and studying the functional roles of genes in organisms.

There are several benefits offered by recombinase-mediated excision over traditional genomic engineering methods, which include:

• The removal of unwanted DNA.
• Recycling of selectable markers.
• Resolution of multiple transgenes to single copy stable sites—preferred in labs because of the simplicity of structural characterization.

Here’re some applications in which recombinases play an essential role:

• Recombinases are involved in improving the specificity of genome modification. It provides a targeted approach for excision, inversion, and insertion events in organisms’ genomes.

For example, recombinases have been used in labs for targeted chromosomal integration, by identifying endogenous genome located recognition sites (or cryptic sites).

• Site-specific recombination is one of the first techniques used to develop transgenic plants involving selectable marker transgene. Several strategies for deleting markers using recombinase have been reported in model plants.
• Apart from transgenic plants, the technique has been used to develop transgenic mammalian cell types, yeast, mice, and Drosophila.

For example, phiC31 integrase (a type of recombinase enzyme) has been used to develop transgenic Xenopus laevis (African clawed frog) embryos. Moreover, the introduction of Cre recombinase RNA in mouse oocytes has been found to induce site-specific recombination of a transgene.

• The site-specific recombinases can be used in many genome engineering applications. They are likely to be used on a routine basis to develop crops that require less pesticide and fertilizers for their healthy growth with increased productivity and quality of foods.
• The site-specific recombinase technology has been a part of genetic modification studies to unravel the answers to fundamental questions in biology and develop biotech tools.
• Recombinases are used to develop artificial inducible recombinase systems in labs for genome engineering experiments or to modify the functions of a gene.

For example, Cre is used to fuse with estrogen hormone receptors, which are involved in inhibiting recombination events, to activate the auto-excision strategy.

• The use of recombinase enzymes in gene therapy has provided a significant step forward for the treatment of incurable diseases through programmed genetics.

Types of Recombinase and Examples

Recombinases are grouped into two families based on their mechanism relatedness and active amino acid within a catalytic domain: serine recombinase family and tyrosine recombinase family.

Tyrosine Recombinases

It has tyrosine amino acid in the catalytic domain that’s involved in DNA attacks and strand exchange processes. The tyrosine recombinase is further divided into two other groups based on the factor if the members utilize identical or non-identical sites.

• Unidirectional recombinases: They utilize non-identical sites and have irreversible inversion, excision, integration, and inversion recombinase activity. Examples are lambda, HK101, pSAM2 recombinases.
• Bidirectional recombinases: They utilize identical sites and their actions are reversible. Their examples are Cre, FLP, and R recombinase.

Serine Recombinases

These recombinases have serine in their catalytic domain site. They are further categorized into two groups based on the size of the enzyme.

• Small recombinases: They are small in size and have irreversible intra-molecular excision actions. The members of this group include β-six, γδ, CinH, and ParA with six, res, RS2, and MRS as their recognition sites.
• Large recombinases: They are large in size and act on non-identical recognition sites—that differ in sequences—known as attP and attB. They perform irreversible inversion, excision, and integration reactions. A reversible action is possible only after the addition of a second protein, known as the corresponding excisionase.

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Figure: An illustration of recombinase superfamily and its members.

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Recombinases are a family of enzymes having functional roles in homologous and site-specific recombination. It’s an event in organisms that involve DNA breakage, strand exchange between homologous segments, and ligation of DNA segments using DNA ligase.

Recombinases are of two types based on common amino acids in the catalytic domain: serine and tyrosine recombinase. These enzymes have several roles in organisms ranging from replication, pathogenesis, to escape host immune responses.

Recombinases were first derived from phages and fungi, and today they are an integral part of genetic engineering applications in labs including genome modification through targeted integration or deletion of a gene segment.

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