User:Emw/Homologous recombination

Homologous recombination, also known as general recombination, is a type of genetic recombination in which genetic material is exchanged between two similar or identical strands of DNA. The process involves several steps of physical breaking and rejoining 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 produce genetic variation (e.g. new, possibly beneficial combinations of alleles) in populations as they reproduce, allowing them to evolutionarily adapt to changing environmental conditions over time.

There are two different types of homologous recombination, one typically involved in DNA repair during mitosis and another involved in meiosis. Both share the same initial steps: after a double-strand break occurs, sections of DNA around the break on the 5' end of the damaged chromosome are removed in a process called resection. In the strand invasion step that follows, an overhanging 3' end of the damaged chromosome then "invades" an undamaged homologous chromosome. A Holliday junction is formed between the two chromosomes after strand invasion. In the DNA repair pathway, a second Holliday junction forms. Depending on how the two junctions are resolved (i.e., cut), the mitotic version results in either chromosomal crossover or non-crossover.

Homologous recombination is widely conserved across all three  domains of life, suggesting that it is a fundamental mechanism in biology. The discovery of genes for homologous recombination in protists has been interpreted as evidence for an early eukaryotic origin of meiosis. Since their dysfunction has been strongly associated with increased susceptibility to several types of cancer, the proteins that facilitate homologous recombination are topics of active research. Homologous recombination is also used as a technique in molecular biology for introducing genetic changes into target organisms. The development of gene targeting techniques that rely on homologous recombination was the subject of the 2007 Nobel Prize for Physiology or Medicine.

Evolutionary origins
Based on the similarity of their amino acid sequences, sets of proteins involved in homologous recombination (HR) are thought to share common evolutionary origins. One such set of HR-related proteins is the recA/RAD51 protein family, which includes the bacterial 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.

Phylogenetic trees indicate that RADA, RAD51 and DMC1 are members of the same monophyletic group, implying that they share a common ancestor. 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. One of the bases for grouping these three proteins together is that they all possess a modified helix-turn-helix motif, which confers DNA-binding activity, toward their N-terminal ends.

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, 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.



RecBCD pathway
The RecBCD pathway is the main homologous recombination pathway used in bacteria to repair double-strand breaks in DNA. After encountering a double-strand break, the RecBCD enzyme binds to the damaged DNA duplex and resects (cleaves off) DNA until it reaches a specific DNA sequence (5'-GCTGGTGG-3') known as a Chi site. RecBCD stops resecting at the Chi site, and then loads multiple RecA enzymes onto the 3' tail (which contains the Chi site) to form a single-strand 3' overhang on each side of the double-strand break. This 3' overhang is a nucleoprotein filament of many RecA proteins bound to a 3' single-stranded DNA tail, with the Chi site at the end of the tail.

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.

It is also used to restart DNA replication stalled by collapsed replication forks, and to regulate gene expression (as in the function of transposons).

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, 1,000–2,000 base pair regions of chromosomes that have high rates of recombination. The absence of a recombination hotspot between two genes on the same chromosome often implies that those genes will be inherited by future generations in equal proportion—that is, there will be higher linkage between the two genes than that expected from  independently assorting genes. 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 result in either crossover or non-crossover in recombinant chromosomes, several genetics studies have suggested the DSBR pathway result predominantly in crossover recombination. Because the DBSR pathway often results in chromosomal crossover, it is the primary model of how homologous recombination occurs during meiosis.

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). Crossover products are also referred to as "splice" products, while non-crossover products are called "patch" products.

SDSA pathway
Homologous recombination via the SDSA pathway occurs in mitosis and results 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.

BIR pathway
During DNA replication, double-strand breaks can sometimes be encountered at replication forks as DNA helicase unzips the template strand. These defects are repaired in the break-induced replication (BIR) pathway of homologous recombination.

The BIR pathway can also help to maintain the length of telomeres, regions of DNA at the end of eukaryotic chromosomes, in the absence of (or in cooperation with) telomerase. Without working copies of the telomerase enzyme, telomeres typically shorten with each round of mitosis, which eventually blocks cell division and leads to senescence. In budding yeast cells where telomerase have been inactivated through mutations, two types of "survivor" cells have been observed to avoid senescence longer than expected by elongating their telomeres through BIR pathways. This recombinatorial telomere elongation occurs through a "roll and spread" mechanism.

The precise molecular mechanisms of the BIR pathway remain unclear. Three proposed mechanisms have strand invasion as an initial step, but differ in how they model the migration of the D-loop and later post-synaptic phases.

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.