Let us make an in-depth study of the genetic recombination. After reading this article you will learn about: 1. Bacterial Recombination 2. Holliday Junction Model 3. Two Holliday Junctions 4. Enzymes of Homologous Recombination and 5. Role of Rec A protein in Homologous Genetic Recombination.
Introduction to Genetic Recombination:
Recombination of DNA takes place by mutation, exchange of DNA strands and incorporation of DNA. In this process the genetic information is rearranged between chromosomes that possess similar sequences. Homologous genetic recombination occurs in eukaryotes at the time of gamete formation during long prophase I of meiosis.
Each chromosome has two sister chromatids, each of which contains a duplex DNA. The homologous chromosomes (one maternal and the other paternal) pair with each other, pairing is known as synapsis and involves entire length of homologous chromosomes.
Recombination occurs by crossing over. It involves reciprocal exchange of chromosomal segments between non-sister chromatids of a homologous pair involving breakage and subsequent reunion in a new arrangement. Chiasma is formed at the site of crossing over. Enzymes like helicases, endonucleases and ligases are involved.
The genetic recombination causes re-arrangement of genes producing altogether new genotypes and phenotypes. These cause variations which lead to evolution. In humans about 30 homologous recombination events occur during each meiosis. The recombination events are much more in bacteria and even more in fungi.
The study of meiosis in lily plants by Herbert Stern and Yasuo Hotta have provided clinching evidence of recombination. Meiocytes of lily flower buds divide synchronously. It has also been discovered that endonuclease, DNA polymerase, ligase and other repair enzymes are present in early prophase.
Mechanics of molecular level of exchange is studied in detail in bacteria and phages. At present we will restrict our discussion to the recombination mechanism where DNA strands recognize each other by complementary strands bounded by base pairs. In bacteria, genetic recombination occurs during conjugation, transformation, transduction post-replication repair, during repair of double strand breaks in DNA, integration of phage DNA with chromosomal DNA and transposons etc. Homologous recombination can lead to gene conversion.
Bacteria are haploid, therefore do not undergo meiosis. They possess only one double stranded DNA molecule or chromosome. There are several types of genetic recombination in microorganisms. The most common recombination is the reciprocal exchange between homologous DNA sequences.
During genetic recombination usually only a part of the genetic material of a donor cell is transferred to a recipient cell. The DNA of the recipient cell and the donor pair with each other and reciprocally exchange DNA strands by crossing over. This gives rise to a new genetic constitution of the recipient cell with new characters. Subsequent daughter cells that contain only recombined chromosome.
There are following main methods by which recombination of genetic material takes place in bacteria.
1. Homologous Recombination:
DNA of the donor cell recombines with recipient DNA by reciprocal exchange of DNA strands. The recombining DNA molecules have homologus sequences. The DNA molecules align or pair with each other and undergo crossing over and homologous recombination. The recombinant DNA has new genetic constitution.
Bacteria can acquire DNA of other closely related bacterial species and can become transformed. This is known as transformation. Transformation was first demonstrated by Griffith in Diploccous pneumoniae bacteria to confirm DNA as genetic material. Homologous recombination is catalysed by rec A protein.
2. Non-homologous Recombination:
Recombination between DNA segments which have no homologous regions is also quite frequent. Here the addition or insertion of a small DNA sequence in a recipient DNA takes place. It occurs mainly by jumping genes or transposons. These mobile DNA sequences regularly jump to a new location anywhere on the genome. Often the transposable elements replicate to generate two copies. One copy remains at the original site while the other jumps to the target site.
3. Site-specific Recombination:
When phage λ (lambda) infects E. coli bacterium, DNA of the phage λ is inserted into the DNA of the bacterium. In this way phage DNA becomes integrated into host DNA and becomes a part of the host chromosome. Bacterium containing a complete set of phage DNA is called lysogen. Phage DNA is inserted at a specific site into the host DNA.
The attachment site of both DNAs possesses identical 15 base pair sequence. A phage encoded protein called integrase catalyses the integration. A host protein called integration host factor (IGF) is also involved. Integrase is a topoisomerase enzyme which breaks, rotates and then re-joins the strands of both molecules. In eukaryotes site-specific recombination produces antibody diversity.
Holliday Junction Model:
Various models to explain homologous genetic recombination have been proposed based primarily on genetic observation in bacteria and fungi. Now it is known that some amount of DNA synthesis also takes place during recombination. DNA replication and repair enzymes are present at the time of genetic recombination.
In 1964 Robin Holliday proposed a model to explain the molecular process involved during the exchange of DNA between two homologous double stranded DNA molecules.
The key steps of this model are as follows:
First of all, pairing or alignment or synapsis between two homologous DNA duplexes takes place. Their sequences are perfectly indentical except that they may contain small regions of different genes called alleles.
Then breaks or nick occur at identical sites in one DNA strand of both homologous DNA duplexes precisely at the same point. The broken ends of strands then invade the opposite complementary strands creating short heteroduplex regions (because of different alleles). This process is called strand invasion. This crossover structure formed is called Holiday junction.
Holliday junction moves in lateral direction. During this process a DNA strand is progressively exchanged with a DNA strand of the other helix. This lateral migration of Holliday junction is called branch migration. The original base pairs are broken in parental molecules and new base pairs are formed in recombined strands.
If the two moelcuels have alternate alleles, A/a, B/b and C/c then the exchange of DNA strands during branch migration produces double strand regions which are not identical. These mismatched regious are called heteroduplexes. During branch migration, the heteroduplex region is elongated.
Breakage and subsequent reunion lead to formation of this joint molecule composed of four interlocked strands of DNA.
The key feature of Holliday junction is the cleavage or cutting across the crossover point which resolves or separates the recombined molecules. To expose two cut sites, the Holliday junction is rotated by 180° to form a sequare planar structure. Resolution of Holliday junction occurs by cutting the DNA strands at the site of cross and re-joining them.
Resolution occurs in one of the two ways. This gives rise to two classes of DNA products. The cut site 1 cleaves the two strands which were initially broken at the start of recombination process (Invading strands are cut). The resolution produces two non-recombinant molecules as only exchange of alleles has taken place in the middle region of duplex B/b and b/B. Thus a patch of hybrid DNA is formed. These molecules are known as patch products.
The cut site 2 cleaves (Non-invading strands are cut) and re-joins two duplexes in such a way that flanking or peripheral genes are exchanged. Here DNA is reciprocally recombined. Crossing over occurs between A and C genes.
Single Strand Exchange Recombination:
According to Mesclson and Radding, a single strand break in one of the two homolgous DNA molecules is quite common.
The free end of the broken strand invades the unbroken double helix and displaces one strand. Rec A protein has the ability to pull out a single strand and displace it. Exonuplease degrades the displaced single strand DNA loop. The unbroken strand of the first molecule serves as a template to synthesize the new strand.
Two Holliday Junctions:
Double stranded breaks in both strands of one DNA molecules occur quite frequently. During DNA repair of double stranded breaks, homologous recombination’s occurs. This type of genetic recombination is called double stranded break repair mechanism. These types of breaks may be caused by ionizing radiations and various damaging agents.
Degeneration of broken strands and generation of single strand tails (ss tails) with 3 tends.
Enzymes Rec BCD further degrades the broken DNA strands. It generates single stranded tails with 3′ ends. One 3′ end tail invades the other unbroken homologous DNA molecule, displacing one of the strands. The invading 3′ end serves as a primer for new DNA synthesis. The displaced strand serves as a template to fill the gap left in the first DNA. If different alleles are present at the site of break, they are permanently lost as regeneration involves homologous DNA as template which has different alleles. This process is called gene conversion because genes of the broken strand are replaced by genes of the homologous DNA.
In double strand break repair, two Holliday junctions are created. These Holliday junctions move laterally by branch migration and the cleavage resolves and separates the two DNA molecules forming a crossover and a non-crossover structure. This is similar to the resolution in the single Holliday junction.
Enzymes of Homologous Recombination:
There are various proteins that catalyse various steps in the process of homologous recombination in E. coli.
Enzymes Rec BCD load onto one end of DNA of double stranded break and move along DNA. (Rec-recombination). In the process it unwinds DNA (helical activity) and degrades one or both DNA strands (nuclease activity). Rec BCD is an endonuclease enzyme.
It is encoded by three genes, Rec B, Rec C and Rec D. Rec BCD continues its degrading activity until it reaches a chi site (%). At this point activities of Rec BCD are stopped. The chi site has eight nucleotides. 5′ GCTGGTGG 3′. The chi sites promote recombination.
The single strand DNA tails generated by BCD enzymes are coated by Rec A enzyme. Rec A stimulates pairing or synopsis between two homologous DNA molecules Rec A also promotes strand invasion, displacing one strand of unbroken DNA molecule and forming D- loop. The displaced strand invades the broken DNA molecule. The missing portions of DNA strands are synthesized using homologous strand as template and gaps are sealed by ligase enzyme.
Ruv AB enzymes recognize and bind to Holliday junction and performs branch migration. Ruv C enzyme cuts DNA strands at Holliday junction and causes separation and resolution of Holliday junction.
Different biological processes like replication, recombination and repair occur in a coordinated manner. In this way new DNA can be synthesized, damaged DNA repaired and genetic recombination takes place. Nucleotide sequences can be replaced through heteroduplexes and gene conversion.
Role of Rec A protein in Homologous Genetic Recombination:
In the various homologous genetic recombination models, the central features are similar in all recombination models.
These include breaks or nicks in DNA molecules. Alignment or pairing or synapsis of homologous sequences of two different DNA molecules. Formation of a crossover structure or Holliday junction in which DNA strand from each molecule creates short regions of heteroduplux DNA. Extension of heteroduplex DNA, which is called branched migration Lastly, resolution of crossover junction to yield end products.
This is an extremely complex process involving the action of several different enzymes. The first event of creating breaks or micks in DNA strands and the last event of resolution are undertaken by various enzymes like helicase, nuclease, and ligases.
But the event starting from pairing of DNA molecules, formation of Holliday junction branch migration are the central features in recombination process. These events are undertaken by a special protein called Rec A protein. Rec A protein is involved in pairing, exchange of strands and branch migration. It is also known as strand exchange protein.
Rec A protein plays a major role in homologous recombination. It is a special protein a completely distinct class of enzymes.
Rec A protein binds quickly to single stranded DNA along the phosphate backbone of DNA helix. DNA is completely covered by Rec A protein. Alongside rec A, a second protein called single strand binding protein (SSB protein) is also involved. Each Rec A molecule has 352 amino acids. There is one rec A monomer every 3-4 nucleotides, of DNA.
Then, the ssDNA in duplex is aligned with homologous sequence of the other DNA molecule. Several steps occur in this process. Two types of homologous interactions occur. The first is the formation of paranemic joints in aligned homologous strands. The end second interaction involves formation of plactonemic joints.
Rec A protein is a DNA dependent ATPase. ATP hydrolysis is required for branch migration, in which strands are replaced and strand exchange occurs. It exhibits polarity as branch migration proceeds in 5′ -> 3′ direction only.
Exchange of DNA Strands:
Role of strand exchange in post-replication repair is very prominent in E. Coli. Strand exchange plays a very prominent, role in repair of DNA damage. As the advancing replication fork comes across a lesion or damaged site such as thymine dimers, it is bypassed during replication process. The damaged protein may be cleaved which may prove to be lethal.
Repair of this lesion requires conversion of this DNA into double stranded DNA and this is achieved by rec A protein. Rec A protein plays its role in retrieving a portion of the complementary strand from other side of the replication fork to fill the gap. This involves branch migration by Rec A protein. This proves that branch migration is essential activity of the cell.