Homologous recombination

[2] Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.

Although homologous recombination varies widely among different organisms and cell types, for double-stranded DNA (dsDNA) most forms involve the same basic steps.

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.

For their development of this technique, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine; Capecchi[3] and Smithies[4] independently discovered applications to mouse embryonic stem cells, however the highly conserved mechanisms underlying the DSB repair model, including uniform homologous integration of transformed DNA (gene therapy), were first shown in plasmid experiments by Orr-Weaver, Szostak and Rothstein.

[5][6][7] Researching the plasmid-induced DSB, using γ-irradiation[8] in the 1970s-1980s, led to later experiments using endonucleases (e.g. I-SceI) to cut chromosomes for genetic engineering of mammalian cells, where nonhomologous recombination is more frequent than in yeast.

[13][15] In 1947, the microbiologist Joshua Lederberg showed that bacteria—which had been assumed to reproduce only asexually through binary fission—are capable of genetic recombination, which is more similar to sexual reproduction.

This work established E. coli as a model organism in genetics,[16] and helped Lederberg win the 1958 Nobel Prize in Physiology or Medicine.

[18][7] During the next decade, experiments in Drosophila, budding yeast and mammalian cells led to the emergence of other models of homologous recombination, called SDSA pathways, which do not always rely on Holliday junctions.

[18] Much of the later work identifying proteins involved in the process and determining their mechanisms has been performed by a number of individuals including James Haber, Patrick Sung, Stephen Kowalczykowski, and others.

[30] Cyclin-dependent kinases (CDKs), which modify the activity of other proteins by adding phosphate groups to (that is, phosphorylating) them, are important regulators of homologous recombination in eukaryotes.

[38] γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute.

[41] At these sites, another protein, SPO11 catalyses recombination-initiating double strand breaks (DSBs), a subset of which are repaired by recombination with the homologous chromosome.

After finding such a sequence, the single-stranded nucleoprotein filament moves into (invades) the similar or identical recipient DNA duplex in a process called strand invasion.

[55] 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 enzyme telomerase, telomeres typically shorten with each cycle of mitosis, which eventually blocks cell division and leads to senescence.

However, in several types of human cancer, a BIR-like pathway helps to sustain some tumors by acting as an alternative mechanism of telomere maintenance.

Upon finding such a sequence, the single-stranded nucleoprotein filament moves into the homologous recipient DNA duplex in a process called strand invasion.

Two sets of the RuvB protein, which each form a ring-shaped ATPase, are loaded onto opposite sides of the Holliday junction, where they act as twin pumps that provide the force for branch migration.

[77][78] In the resolution phase of recombination, any Holliday junctions formed by the strand invasion process are cut, thereby restoring two separate DNA molecules.

[80] Studies in several species of bacteria have established that there is a log-linear decrease in recombination frequency with increasing difference in sequence between host and recipient DNA.

In order for a bacterium to bind, take up and integrate donor DNA into its resident chromosome by homologous recombination, it must first enter a special physiological state termed competence.

The findings indicate that the 11083G > T mutation of SARS-CoV-2 spread during shipboard quarantine and arose through de novo RNA recombination under positive selection pressure.

In addition, in three patients in this cruise, two mutations 29736G > T and 29751G > T ("G13" and "G28") were also located in Coronavirus 3′ stem-loop II-like motif (s2m), as "G28" was predicted as recombination hotspots in Australian SARS-CoV-2 mutants.

[104] In the cells of Bloom's syndrome patients, who lack a working copy of the BLM protein, there is an elevated rate of homologous recombination.

[105] Experiments in mice deficient in BLM have suggested that the mutation gives rise to cancer through a loss of heterozygosity caused by increased homologous recombination.

In recognition of their discovery of how homologous recombination can be used to introduce genetic modifications in mice through embryonic stem cells, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine.

[118] Advances in gene targeting technologies which hijack the homologous recombination mechanics of cells are now leading to the development of a new wave of more accurate, isogenic human disease models.

These techniques exploit the fact that recombination can introduce a high degree of sequence diversity while preserving a protein's ability to fold into its tertiary structure, or three-dimensional shape.

[121] In a study published in 2007, researchers were able to create chimeras of two enzymes involved in the biosynthesis of isoprenoids, a diverse class of compounds including hormones, visual pigments and certain pheromones.

[128][129] Olaparib, a PARP1 inhibitor, shrunk or stopped the growth of tumors from breast, ovarian and prostate cancers caused by mutations in the BRCA1 or BRCA2 genes, which are necessary for HR.

Depiction of chromosome 1 after undergoing homologous recombination in meiosis
Figure 1. During meiosis , homologous recombination can produce new combinations of genes as shown here between similar but not identical copies of human chromosome 1 .
Figure 2. An early illustration of crossing over from Thomas Hunt Morgan
Figure 3. Homologous recombination repair attempts occur in DNA before the cell enters mitosis (M phase shown) during the S and G 2 phases of the cell cycle .
Figure 4. Double-strand break repair models that act via homologous recombination
See adjacent text.
Figure 5. The DSBR and SDSA pathways follow the same initial steps, but diverge thereafter. The DSBR pathway most often results in chromosomal crossover (bottom left), while SDSA always ends with non-crossover products (bottom right).
Still frame of an animation of the SSA pathway. A single molecule of double-stranded DNA is shown in red, oriented horizontally. On each of the two DNA strands, two purple regions indicating repeat sequences of DNA are shown to the left and right of the center of the DNA molecule.
Figure 6. Recombination via the SSA pathway occurs between two repeat elements (purple) on the same DNA duplex, and results in deletions of genetic material. (Click to view animated diagram in Firefox , Chrome , Safari , or Opera web browsers.)
Crystal structure of RecA bound to DNA.
Figure 7. Crystal structure of a RecA protein filament bound to DNA. [ 58 ] A 3' overhang is visible to the right of center.
Figure 8A. Molecular model for the RecBCD pathway of recombination. This model is based on reactions of DNA and RecBCD with ATP in excess over Mg2+ ions. Step 1: RecBCD binds to a double-stranded DNA end. Step 2: RecBCD unwinds DNA. RecD is a fast helicase on the 5’-ended strand, and RecB is a slower helicase on the 3'-ended strand (that with an arrowhead) [ref 46 in current Wiki version]. This produces two single-stranded (ss) DNA tails and one ss loop. The loop and tails enlarge as RecBCD moves along the DNA. Step 3: The two tails anneal to produce a second ss DNA loop, and both loops move and grow. Step 4: Upon reaching the Chi hotspot sequence (5' GCTGGTGG 3'; red dot) RecBCD nicks the 3’-ended strand. Further unwinding produces a long 3'-ended ss tail with Chi near its end. Step 5: RecBCD loads RecA protein onto the Chi tail. At some undetermined point, the RecBCD subunits disassemble. Step 6: The RecA-ssDNA complex invades an intact homologous duplex DNA to produce a D-loop, which can be resolved into intact, recombinant DNA in two ways. Step 7: The D-loop is cut and anneals with the gap in the first DNA to produce a Holliday junction. Resolution of the Holliday junction (cutting, swapping of strands, and ligation) at the open arrowheads by some combination of RuvABC and RecG produces two recombinants of reciprocal type. Step 8: The 3' end of the Chi tail primes DNA synthesis, from which a replication fork can be generated. Resolution of the fork at the open arrowheads produces one recombinant (non-reciprocal) DNA, one parental-type DNA, and one DNA fragment. [ 61 ]
See caption.
Figure 8B. Beginning of the RecBCD pathway. This model is based on reactions of DNA and RecBCD with Mg 2+ ions in excess over ATP. Step 1: RecBCD binds to a DNA double strand break. Step 2: RecBCD initiates unwinding of the DNA duplex through ATP-dependent helicase activity. Step 3: RecBCD continues its unwinding and moves down the DNA duplex, cleaving the 3' strand much more frequently than the 5' strand. Step 4: RecBCD encounters a Chi sequence and stops digesting the 3' strand; cleavage of the 5' strand is significantly increased. Step 5: RecBCD loads RecA onto the 3' strand. Step 6: RecBCD unbinds from the DNA duplex, leaving a RecA nucleoprotein filament on the 3' tail. [ 62 ]
Schematic representation of the s2m RNA secondary structure, with tertiary structural interactions indicated as long range contacts.
Figure 9. Joining of single-ended double strand breaks could lead to rearrangements
Graphic showing proteins from each domain of life. Each protein is shown horizontally, with homologous domains on each protein indicated by color.
Figure 10. Protein domains in homologous recombination-related proteins are conserved across the three main groups of life: archaea, bacteria and eukaryotes.
A mouse with a white coat blotched with brown is shown on the right, with two smaller brown mice to the immediate left.
Figure 11. As a developing embryo, this chimeric mouse had the agouti coat color gene introduced into its DNA via gene targeting. Its offspring are homozygous for the agouti gene.