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Recombinase
Recombinase
Recombinase
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Recombinases are genetic recombination enzymes.

Site specific recombinases

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Further information: Site-specific recombinase technology

DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes, and to control gene expression. These enzymes, derived from bacteria (bacteriophages) and fungi, catalyze directionally sensitive DNA exchange reactions between short (30–40 nucleotides) target site sequences that are specific to each recombinase. These reactions enable four basic functional modules: excision/insertion, inversion, translocation and cassette exchange, which have been used individually or combined in a wide range of configurations to control gene expression.[1][2][3][4][5]

Types include:

Homologous recombination

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Recombinases have a central role in homologous recombination in a wide range of organisms. Such recombinases have been described in archaea, bacteria, eukaryotes and viruses.

Archaea

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The archaeon Sulfolobus solfataricus RadA recombinase catalyzes DNA pairing and strand exchange, central steps in recombinational repair.[6] The RadA recombinase has greater similarity to the eukaryotic Rad51 recombinase than to the bacterial RecA recombinase.[6]

Bacteria

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RecA recombinase appears to be universally present in bacteria. RecA has multiple functions, all related to DNA repair. RecA has a central role in the repair of replication forks stalled by DNA damage and in the bacterial sexual process of natural genetic transformation.[7][8]

Eukaryotes

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Eukaryotic Rad51 and its related family members are homologous to the archaeal RadA and bacterial RecA recombinases. Rad51 is highly conserved from yeast to humans. It has a key function in the recombinational repair of DNA damages, particularly double-strand damages such as double-strand breaks. In humans, over- or under-expression of Rad51 occurs in a wide variety of cancers.

During meiosis Rad51 interacts with another recombinase, Dmc1, to form a presynaptic filament that is an intermediate in homologous recombination.[9] Dmc1 function appears to be limited to meiotic recombination. Like Rad51, Dmc1 is homologous to bacterial RecA.

Viruses

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Some DNA viruses encode a recombinase that facilitates homologous recombination. A well-studied example is the UvsX recombinase encoded by bacteriophage T4.[10] UvsX is homologous to bacterial RecA. UvsX, like RecA, can facilitate the assimilation of linear single-stranded DNA into an homologous DNA duplex to produce a D-loop.

References

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Recombinase

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from Grokipedia
Recombinases are a family of enzymes that catalyze the recombination of DNA molecules, enabling the exchange, integration, excision, or inversion of genetic segments to maintain genome integrity, facilitate DNA repair, and generate genetic diversity. These enzymes play critical roles in biological processes ranging from homologous recombination during DNA damage repair to site-specific rearrangements in viral integration and gene regulation. In homologous recombination, recombinases such as RecA in bacteria and Rad51 or Dmc1 in eukaryotes form nucleoprotein filaments on single-stranded DNA, promoting strand invasion and exchange between homologous DNA sequences to repair double-strand breaks or support meiotic crossing over. This process ensures high-fidelity repair by using a sister chromatid or homologous chromosome as a template, preventing mutations and preserving genetic information. Site-specific recombinases, by contrast, recognize short, defined DNA sequences (such as loxP or FRT sites) without requiring extensive homology, allowing precise genomic modifications like the integration of transgenes or removal of selectable markers. These enzymes are classified into tyrosine recombinases (e.g., Cre from bacteriophage P1, which mediates bidirectional recombination) and serine recombinases (e.g., phiC31 integrase, often unidirectional and used for stable insertions), and they have revolutionized genetic engineering in model organisms, plants, and therapeutic applications. Beyond their natural roles in cellular maintenance and , recombinases have broad applications in , including the creation of conditional knockouts in mice via Cre-lox systems, marker-free transgenic crops, and emerging gene therapies for diseases like cancer and genetic disorders, highlighting their versatility and precision in manipulating genomes.

Overview

Definition and General Function

Recombinases are a class of enzymes that catalyze the exchange of genetic information between DNA molecules, facilitating rearrangements such as the breaking and rejoining of DNA strands at specific or homologous sites. These enzymes play essential roles in processes including the integration of viral or into host genomes, the excision of such elements, the inversion of DNA segments to alter gene orientation, and the repair of double-strand breaks in DNA. Recombinase-mediated recombination can result in conservative or replicative outcomes. In conservative recombination, typically associated with site-specific mechanisms, there is no net gain or loss of DNA nucleotides, as the process involves precise breakage and rejoining without replication. In contrast, replicative recombination incorporates DNA synthesis, often leading to the duplication of genetic elements, such as in certain transposition events. Many recombinases require cofactors like ATP to drive strand invasion and exchange, particularly in homology-directed processes, while others operate without such energy inputs. Overall, these enzymes are vital for maintaining stability by enabling accurate and preventing chromosomal aberrations during replication and .

Biological and Evolutionary Significance

Recombinases are evolutionarily conserved across all three domains of life—, , and Eukarya—indicating their ancient origins likely traceable to the (LUCA). machinery, including RecA-like proteins (such as RadA in and Rad51 in Eukarya), facilitates and genetic exchange in diverse organisms, suggesting this system was present in the primordial cellular community from which modern life diverged. Similarly, site-specific recombinases, including and serine families, are ubiquitous, enabling precise DNA rearrangements that underpin fundamental cellular processes from prokaryotic maintenance to eukaryotic development. This broad conservation underscores recombinases' role as foundational enzymes in maintaining integrity amid environmental pressures since life's early evolution. Recombinases drive by promoting (HGT), which accelerates and contributes to phenomena like antibiotic resistance dissemination and bacterial . Site-specific recombinases, such as integrases, integrate (e.g., integrative conjugative elements and prophages) into bacterial chromosomes, often carrying antibiotic resistance genes that spread rapidly across populations via conjugation or transduction. For instance, over 90% of such elements target conserved hotspots near tRNA genes, minimizing host fitness costs while enabling the acquisition of adaptive traits. In evolutionary contexts, fosters interspecies genetic exchange, enhancing allelic diversity and potentially driving incipient by generating novel genotypes that promote ecological divergence, as seen in mismatch repair mutants that elevate recombination rates. These enzymes contribute to genome plasticity, allowing organisms to respond dynamically to selective pressures through mechanisms like phase variation in pathogens and V(D)J recombination for immune diversity. In bacteria, site-specific recombinases mediate reversible DNA inversions that toggle expression of virulence factors, such as adhesins or toxins, enabling pathogens like Escherichia coli to evade host immunity and colonize diverse niches. In eukaryotes, RAG1 and RAG2 recombinases orchestrate V(D)J recombination in lymphocytes, assembling diverse immunoglobulin and T-cell receptor genes to generate trillions of antigen-specific clones essential for adaptive immunity. Dysregulation of recombinases links to diseases: excessive homologous recombination promotes genomic instability in cancers like multiple myeloma, while defects in RAG cause severe combined immunodeficiency (SCID) and increase leukemia risk through off-target cleavages. Similarly, impaired homologous recombination underlies genetic disorders like Bloom's syndrome, characterized by heightened cancer predisposition due to unrepaired DNA breaks.

Classification of Recombinases

Site-Specific Recombinases

Site-specific recombinases are highly specialized enzymes that catalyze precise DNA rearrangements, such as integration, excision, inversion, or resolution, by recombining DNA segments at short, specific recognition sequences typically 20–40 base pairs in length. Unlike , these enzymes operate independently of extensive sequence homology between the participating DNA molecules, relying instead on the exact matching of their defined target sites. This sequence specificity ensures high-fidelity modifications, often forming transient intermediates like Holliday junctions in certain families, and enables directional outcomes based on the orientation of the recognition sites. Site-specific recombinases are broadly classified into two major families based on their catalytic nucleophiles and mechanisms: recombinases and serine recombinases. The recombinase family, also encompassing the lambda integrase subfamily, uses a conserved residue to initiate DNA cleavage, breaking and rejoining strands in a pairwise manner that generates intermediates. These enzymes require directional sites, such as the attP (phage) and attB (bacterial) sequences in lambda integrase-mediated recombination, where accessory proteins like integration host factor (IHF) and excisionase (Xis) regulate the directionality for integration or excision of phage DNA into the host genome. Representative examples include Cre from bacteriophage P1, which recombines at loxP sites (34 bp) for resolution or , and FLP from the 2-micron , which acts on FRT sites (34 bp) to mediate maintenance. In contrast, serine recombinases employ a serine residue as the , performing a concerted cleavage of all four DNA strands before strand exchange, without forming Holliday junctions and often altering central dinucleotide sequences in the process. This family is divided into small and large serine recombinases. Small serine recombinases include resolvases and invertases, with resolvases such as γδ from the Tn1000 transposon or those from Tn3 resolving multimeric forms into monomers by recombining directly repeated res sites (approximately 114 , comprising subsites I, II, and III), thereby promoting stable segregation during . These enzymes typically feature an N-terminal catalytic domain and a C-terminal , ensuring strict specificity at the crossover region flanked by inverted repeats. Large serine recombinases, such as the φC31 integrase from , mediate primarily unidirectional integration of DNA at asymmetric attachment (att) sites, often requiring recombination directionality factors (RDFs) for excision; they possess an N-terminal catalytic domain and a complex C-terminal multidomain region for enhanced specificity and regulation.

Homologous Recombinases

Homologous recombinases are a class of enzymes that catalyze the exchange of genetic material between DNA molecules sharing , typically to repair double-strand breaks or facilitate during . Unlike site-specific recombinases, these proteins rely on extended regions of sequence similarity rather than defined recognition sites, enabling accurate template-directed repair or allelic exchange. This process is conserved across domains of life and is essential for maintaining genomic stability in response to DNA damage. The core family of homologous recombinases comprises RecA-like proteins, which share structural and functional homology. In bacteria such as , RecA forms the foundational recombinase, binding cooperatively to single-stranded (ssDNA) to create a nucleoprotein filament that searches for and invades homologous duplex . Eukaryotic counterparts include Rad51, which performs similar roles in mitotic and meiotic cells for , and the meiosis-specific Dmc1, which shares approximately 50% identity with Rad51 and specializes in chromosome pairing during . These proteins exhibit ATP-dependent activity, driving filament assembly and disassembly. Key mechanistic features of homologous recombinases involve the formation of extended helical filaments on ssDNA, which extend and stiffen the DNA to facilitate homology recognition and strand . This filament structure promotes the exchange of DNA strands, forming displacement loops (D-loops) that initiate recombination. Accessory proteins enhance efficiency; for instance, in , the RuvAB complex, consisting of the RuvA clamp and RuvB hexameric , drives branch migration of Holliday junctions post-invasion, resolving recombination intermediates. In eukaryotes, mediators like Rad52 and assist Rad51 filament nucleation on RPA-coated ssDNA. Prominent examples illustrate their biological roles. In E. coli, orchestrates the SOS response to DNA damage by forming filaments that not only promote for repair but also induce error-prone translesion synthesis, balancing survival and mutagenesis. In humans, Rad51 is critical for meiotic recombination, where it collaborates with Dmc1 to ensure proper crossover formation and chromosome segregation, preventing . Disruptions in Rad51 function are linked to genomic instability and cancer predisposition, underscoring its high-fidelity repair role.

Mechanisms of Recombination

Site-Specific Recombination Process

Site-specific recombination is a precise DNA rearrangement process mediated by recombinases that recognize short, specific DNA sequences called recombination sites, enabling reactions such as integration, excision, or inversion without requiring extensive homology or DNA synthesis. The process is conservative, deriving energy from the cleavage and reformation of phosphodiester bonds, and proceeds through a series of tightly regulated steps involving protein-DNA complexes. Recombinases are classified into two major families—tyrosine and serine—each employing distinct catalytic mechanisms but sharing the overarching goal of site-specific strand breakage and rejoining. The process begins with synapsis, where recombinase monomers bind to the specific DNA sites, typically forming dimers on each site due to cooperative interactions. These dimers then align and associate into a higher-order synaptic complex, bringing the two recombination sites into close proximity in an antiparallel orientation to facilitate recombination. For example, in the Cre-lox system, two Cre dimers on loxP sites form a tetrameric complex, with DNA bending aiding alignment. In serine recombinases, synapsis similarly involves dimer binding followed by tetramer formation, often stabilized by accessory factors or DNA topology. Cleavage and strand exchange follow, differing between families. In tyrosine recombinases, cleavage occurs sequentially on one pair of strands via nucleophilic attack by a conserved tyrosine residue, forming a covalent 3'-phosphotyrosine intermediate and freeing 5'-hydroxyl ends. The 5'-hydroxyls then attack the phosphotyrosine bonds on the partner site, exchanging strands and generating a Holliday junction intermediate—a four-way DNA structure. Isomerization of this junction activates cleavage on the second pair of strands, completing exchange. Serine recombinases, by contrast, cleave all four strands simultaneously through serine hydroxyl nucleophilic attack, creating 5'-phosphoserine covalent intermediates and 3'-hydroxyl ends, without forming a Holliday junction; strand exchange proceeds via subunit rotation within the synaptic tetramer. Resolution occurs through religation: the free hydroxyls attack the protein-DNA intermediates, reforming phosphodiester bonds and yielding recombinant products, such as fused DNA molecules or catenanes. Directionality—determining whether recombination results in integration, excision, or inversion—is controlled by accessory proteins, , and site architecture. can drive formation and favor specific topologies, while accessory proteins like recombination directionality factors (RDFs) in serine integrases promote excision by altering synaptic interfaces. In tyrosine systems, influences strand selectivity during . The overall reaction can be simplified as: DNA1-site+DNA2-site→DNA1-site-DNA2\text{DNA1-site} + \text{DNA2-site} \rightarrow \text{DNA1-site-DNA2} where the energy is provided by transient breakage and reformation, ensuring no net ATP consumption.

Homologous Recombination Process

(HR) is an ATP-dependent process mediated by homologous recombinases such as RecA in prokaryotes and Rad51 in eukaryotes, which facilitates the repair of double-strand breaks (DSBs) or the exchange of genetic material between homologous DNA sequences. The pathway begins with the processing of DSB ends to generate single-stranded DNA (ssDNA) overhangs, followed by the assembly of a recombinase filament on the ssDNA, homology searching, strand invasion to form intermediates, and final resolution of recombination products. This mechanism ensures accurate repair by using a homologous template, distinguishing it from non-homologous end joining.02158-4)

Presynapsis

Presynapsis initiates HR through the assembly of a nucleoprotein filament on ssDNA generated by resection of DSB ends via nucleases such as in or MRN complex (Mre11-Rad50-Nbs1) in eukaryotes. Recombinases like or Rad51 bind cooperatively to the ssDNA, displacing accessory proteins such as single-stranded DNA-binding protein (SSB) or (RPA), to form an extended helical filament with ~6-7 monomers per DNA turn, stabilized by ATP binding. This filament adopts a right-handed helical structure that stretches the ssDNA by ~1.5-fold, priming it for homology search. The homology search occurs via paranemic joining, an initial non-Watson-Crick base pairing between the ssDNA filament and a double-stranded DNA (dsDNA) donor, allowing rapid sampling of potential homologous sequences without topological entanglement. This ATP-dependent scanning process, facilitated by the filament's dynamic conformational changes, enables efficient alignment over kilobase distances before stable . Accessory proteins like Rad52 or mediate filament nucleation and stability, enhancing search efficiency .

Strand Invasion

Once homology is identified, strand invasion proceeds with the recombinase filament promoting the pairing of the ssDNA with the complementary strand in the homologous dsDNA, forming a intermediate. In this step, the invading ssDNA displaces one strand of the dsDNA, creating a three-stranded stabilized by , which drives unidirectional branch migration to extend the heteroduplex region. For , this invasion is polarity-specific, favoring 5'-to-3' progression along the ssDNA. Subsequent second end capture aligns the other DSB end with the , often mediated by annealing proteins like Rad52, leading to the formation of a double (dHJ) or a single HJ in alternative models. Branch migration continues ATP-dependently, powered by motor proteins such as RuvAB in or BLM in eukaryotes, to enlarge the heteroduplex and resolve potential topological barriers.00850-7) The overall reaction can be represented as: ssDNA (filament-bound)+homologous dsDNA→ATPD-loop intermediate→Holliday junction(s)\text{ssDNA (filament-bound)} + \text{homologous dsDNA} \xrightarrow{\text{ATP}} \text{D-loop intermediate} \to \text{Holliday junction(s)}
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