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Homologous recombination
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Homologous recombination, also known as general recombination, is a type of genetic recombination that involves a genetic exchange between two similar or identical strands of DNA. Although most widely used in cells to accurately repair double-strand breaks in DNA, homologous recombination also produces new combinations of DNA sequences during chromosomal crossover in meiosis. These new combinations of DNA in turn produce genetic variation (e.g.
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Encyclopedia
Homologous recombination, also known as general recombination, is a type of genetic recombination that involves a genetic exchange between two similar or identical strands of DNA. Although most widely used in cells to accurately repair double-strand breaks in DNA, homologous recombination also produces new combinations of DNA sequences during chromosomal crossover in meiosis. These new combinations of DNA in turn produce genetic variation (e.g. new, possibly beneficial combinations of alleles) in populations as they reproduce, allowing them to evolutionarily adapt to environmental conditions over time.
The process of homologous recombination involves the alignment of similar (i.e. homologous) DNA sequences, formation of a Holliday junction, and breaking and repair, known as resolution, of the DNA to produce an exchange of material between the strands. Widely conserved among both prokaryotes and eukaryotes, homologous recombination is also used as a technique in molecular biology for introducing genetic changes into target organisms.
Evolutionary origins
Phylogenetic analyses have used sequence similarity within sets of proteins involved in homologous recombination to derive models for their shared evolutionary origins. One such set of homologous recombination-related proteins is the recA/RAD51 protein family, which includes the prokaryotic recA protein and homologous proteins in archaea (RADA and RADB) and eukaryotes (RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2 and XRCC3). All of these proteins share a conserved region of approximately 230 amino acids in length, known as the recA/RAD51 domain. Within this protein domain are two sequence motifs, Walker A and Walker B, which confer ATP hydrolysis activity to the protein products of all members of the recA/RAD51 gene family.
The archaeal protein RADA and the eukaryotic homologs RAD51 and DMC1 all possess a modified helix-turn-helix (HhH) motif, which confers DNA-binding activity, toward their N-terminal ends. Phylogenetic trees constructed using both the neighbor-joining and maximum likelihood methods indicate that these three genes are members of the same monophyletic group, deemed the RADα family. Within this protein family, RAD51 and DMC1 are grouped together in a separate clade from RADA. An ancient gene duplication event of a eukaryotic RECA gene has been proposed as a likely origin of the modern RAD51 and DMC1 genes.
The discovery of DMC1 in several species of Giardia, one of the earliest protists to diverge as a eukaryote, suggests that meiotic homologous recombination (and thus meiosis itself) emerged very early in eukaryotic evolution.
In addition to research on DMC1, molecular and phylogenetic analyses of the Spo11 protein and its homologs have also provided information on the origins of meiotic recombination. Spo11 is a type II topoisomerase that catalyzes the double-strand breaks necessary to initiate homologous recombination in meiosis. Phylogenetic trees constructed from inferred protein sequences of Spo11 gene homologs in animals, fungi, some plants, and protists and archaea suggest that eukaryotic Spo11 emerged in the last common ancestor of eukaryotes and archaebacteria.
In bacteria
Homologous recombination is a major DNA repair process in bacteria. It is also important for producing genetic diversity in bacterial populations, although the process differs substantially from meiotic recombination, which brings about diversity in eukaryotic genomes. Understanding of homologous recombination is most advanced for Escherichia coli, due to the organism's standing as a model organism in molecular genetics. Two well-known versions of the pathway are the RecBCD pathway, which aids in the repair of double-strand breaks in DNA, and the RecFOR pathway, which promotes repair of single-strand breaks. The RecBCD pathway is used in DNA repair to restart replication forks that have been stalled or damaged, and to regulate gene expression (as in the function of transposons). Additionally, due to recognition of recombination enzymes of specialized sites within the bacterial chromosome, foreign DNA can be degraded, thus protecting the E. coli cell .
In the double strand break repair pathway, homologous recombination is mediated by RecA and RecBCD, along with RuvABC. These enzymes are attracted to double strand breaks, search for sequence similarity between the duplex strands and catalyze formation of a Holliday junction, branch migration, and resolution. RecBCD assembles at the double stranded break, then, by exonuclease activity, cleaves off the DNA until it reaches a chi site. Once this occurs, RecD is inactivated or lost, and the enzyme continues to cut the DNA strand, leaving a 3' tail.
RecA binds to the single-stranded DNA, forming a nucleoprotein filament. RecA protects the DNA from single-strand binding proteins by coating the 3' tail, as it facilitates hybridization between the single-stranded region and the double-stranded DNA, known as strand invasion, needing a region of only about 15 complementary base pairs. Where the strands cross is known as the Holliday junction.
RuvA binds to the Holliday junction and recruits RuvB. Movement of the Holliday junction down the DNA strand, known as branch migration, is catalyzed by RuvB, a hexameric ATPase. RuvC is an endonuclease that cuts with slight specificity, allowing some degree of branch migration before resolving the junction.
There are two types of products resulting from recombination, stemming from resolution of the Holliday junction: splice and patch. Splice products are crossover products, in which there is reshuffling of genes, while patch resolution yields non-crossover products.
In bacteria, homologous recombination introduces DNA into a bacterium through conjugation, transduction, or transformation.
In eukaryotes
Homologous recombination is essential to mitosis and meiosis in most eukaryotic cells. In mitosis, homologous recombination repairs double-strand breaks in DNA caused by ionizing radiation or DNA-damaging chemicals. When left unrepaired, these double-strand breaks can cause large-scale chromosomal rearrangements in somatic cells, which can in turn lead to cancer. In meiosis, homologous recombination facilitates chromosomal crossover during prophase I. Meiotic homologous recombination begins when the Spo11 protein makes a programmed double-strand break in DNA. The sites of these double-strand breaks often occur at recombination hotspots, 1,500–2,000 base pair regions of chromosomes that have high rates of recombination. The shuffling of genetic material between parental chromosomes that results is an important source of genetic diversity in subsequent generations.
Double-strand break repair
The two primary models for double-strand break repair (DSBR) in DNA are the DSBR pathway (sometimes called the double Holliday junction model) and the synthesis-dependent strand annealing (SDSA) pathway. The two pathways are similar in their first several steps. After a double-strand break occurs, both are initiated by a "resection" of the double-strand break, in which DNA immediately upstream (i.e., toward the 5' end) of the double-strand break is removed on each strand of the DNA duplex, leaving two 3' overhangs of single-stranded DNA. Next, in a process called strand invasion, one of these single-stranded overhangs forms a "presynaptic filament" with Rad51 (and Dmc1, in meoisis) and its accessory proteins, which together then moves into (invades) a homologous chromosome – often a sister chromatid of the damaged chromosome. A displacement loop (D-loop) is formed during strand invasion between the invading 3' overhang strand and the homologous chromosome. After strand invasion, a DNA polymerase extends the invading 3' strand, changing the D-loop to more prominently cruciform structure known as a Holliday junction. Following this, DNA synthesis occurs on the invading strand (i.e., one of the original 3' overhangs), effectively restoring the strand on the homologous chromosome that was displaced during strand invasion.
DSBR pathway
After the stages of resection, strand invasion and DNA synthesis outlined above, the DSBR and SDSA pathways become distinct. The DSBR pathway is unique in that the second 3' overhang (which was not involved in strand invasion) also forms a Holliday junction with the homologous chromosome. The double Holliday junctions are then converted into recombination products by nicking endonucleases, a type of restriction endonuclease which only cleaves one DNA strand. While it was thought to results in either crossover or non-crossover in recombinant chromosomes, several genetics studies have suggested the DSBR pathway result predominantly in crossover recombination.
Whether recombination in the DSBR pathway results in chromosomal crossover is determined by how the double Holliday junction is resolved. If the two Holliday junctions are cleaved on the crossing strands (along the black arrowheads at both Holliday junctions in the accompanying figure), then chromosomes without crossover will be produced. Alternatively, chromosomal crossover will occur if one Holliday junction is cleaved on the crossing strand and the other Holliday junction is cleaved on the non-crossing strand (i.e., along the blacks arrowheads at one Holliday junction and along the orange arrowheads at the other in the figure).
SDSA pathway
Homologous recombination via the SDSA pathway occurs in both mitosis and meoisis, resulting in non-crossover (NCO) products. In this model, movement of the Holliday junction down the DNA strand (a process called branch migration) ends with the release of the extended invading strand. The newly synthesized 3' end of the invading strand is then able to anneal to the other original 3' overhang in the damaged chromosome through complementary base pairing. SDSA is completed with the removal of 3' flaps left over after annealing and the ligation of any remaining single-stranded gaps.
Effects of dysfunction
Deficiencies in homologous recombination (HR) have been strongly linked to cancer formation in humans. For example, each of the cancer-related diseases Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome are caused by malfunctioning copies of RecQ helicase genes involved in HR regulation: BLM, WRN and RECQ4, respectively. In the case of Bloom's syndrome patients, who lack a working copy of the BLM protein, cells have an elevated rate of homologous recombination. Experiments done in mice deficient in BLM have suggested that the mutation gives rise to cancer through a loss of heterozygosity caused by increased homologous recombination.
Decreased rates of homologous recombination can also lead to cancer. This is the case with BRCA1, a tumor suppressor genes whose malfunctioning has been prominently associated with increased susceptibility to breast and ovarian cancer. Cells missing BRCA1 were shown to have a five-fold decrease in homologous recombination events and increased sensitivity to ionizing radiation (indicating more unrepaired double-strand breaks in DNA). The reintroduction of BRCA1 saw a simultaneous increase in homologous recombination events and decrease in sensitivity to ionizing radiation. Facilitating homologous recombination is the only known function of a closely-related gene, BRCA2. Its large protein product, the 3418-amino acid long BRCA2 protein, aids homologous recombination by binding to single-stranded DNA and providing a platform for the extension of the RAD51 filament. This filament formation is an important step in the initiation of homologous recombination, and cells made deficient in this process by mutant copies of the BRCA2 protein were shown have a similar phenotype to BRCA1 mutants: decreased homologous recombination and increased sensitivity to radiation.
Uses in biotechnology
Many methods for introducing DNA sequences into organisms to create recombinant DNA and genetically modified organisms use the process of homologous recombination. Also called gene targeting, the method is especially common in yeast and mouse genetics. The gene targeting method in knockout mice uses mouse embryonic stem cells to deliver artificial genetic material (mostly of therapeutic interest), which represses the target gene of the mouse by the principle of homologous recombination. The mouse thereby acts as a working model to understand the effects of a specific mammalian gene. This work yielded Mario Capecchi, Martin Evans and Oliver Smithies the 2007 Nobel Prize for Physiology or Medicine.
See also
External links
: Adobe Flash-based animations showing several models of homologous recombination
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