Recombination
Recombination is the process in which one or more nucleic acid molecule interact with one another to bring about rearrangement of genetic information in an organism and produce new nucleotide sequences. Recombination is accompanied by phenotypic change.
Genetic recombination is of many types. But the two most common type of recombination are:
a) General/Homologus recombination
b) Site specific recombination
General / Homologus Recombination:
It is the most common form of recombination. It usually involves a reciprocal exchange between a pair of homologus DNA sequence. It results from DNA strand breakage and reunion leading to crossover. Homologus reaction is a reaction between two dupleses of DNA. Its critical feature is that the enzymes responsible can use any pair of homologus sequence as substrates. This recombination occurs during the prophase of meiosis. The begining of meiosis is marked by the point at which individual chromosomes becomes visible. Each of these chromosomes have replicated previously and consists of two sister chromatids, each of which contains duplex DNA.
The homologus chromosome approach one another and begin to pair in one or more regions forming bivalents. Pairing extends untill the entire length of each chromosome is apposed with its homolog. the process is called as synopsis or chromosome pairing. When the process is completed, the chromosomes are laterally associated in the form of synaptonemal complexes.
Recombination between chromosomes involves a physical exchange of parts, usually represented as a breakage and union in which two non-sister chromatids are broken and linked each with the other. When the chromosome begin to separate, they can be held together at discrete sites, the chiasmata. The number and distribution of chiasmata parallel the feature of genetic crossing over. The chiasmata remain visible when the chromosomes condense and all four chromosomes becomes evident.
Holiday model for homologus chromosome:
General studies on fungi led Robin Holoday to suggest a protypical model for general recombination in 1964. Recombination begins with the two DNA duplexes lined up in a manner that their homologus sequence are alligned. A single stranded nick is introduced at the same point on the corresponding strands of the homologus DNA duplexes. The nicked ends are displaced to other molecule and base pairs with the complementary sequence on that molecule. DNA-ligase seals the nick on each transplanted strand, creating a heteroduplex molecule from the two homologus.
After initial base pairing, more of each strand is displaced across the other molecule in a zipper like action. This process is called as branch migration, because the branch point between the two molecules moves along the heteroduplex. this structure is called as Holiday Junction. It is only an intermediate and if recombination is to succeed, this structure has to be resolved to create two separate DNA molecules, which is done by rotating one duplex relative to other.
Resolution requires a further pair of nicks. Resolution of holiday junction can generate parental or recombinant duplexes, depending on which strands are nicked. Both the type of products have a region of heteroduplex DNA. If the nicks are made at in the pair of strands that were not originally nicked, all four of the original strands have been nicked. This releases splice recombinant DNA molecules. The duplex of DNA parent is covalently linked to the duplex of other DNA parent via stretch of heteroduplex region. If the same two strands involved in the original nicking are nicked again, the other two strands remain intact. The nicking releases the original parental duplexes, which remain intact except that has a residum of the event in the form of a length of heteroduplex DNA. These are called as patch recombinant.
These alternative resolutions establish the principle that a strand exchange between duplex DNAs always leaves behind a region of heteroduplex DNA, but the exchange may or may not be accompanied by recombing of the fklanking regions. The nicks are sealed by DNA ligase.
Mechanism of recombination:
Recombination is initiated by an endonuclease, that cleaves one of the partner DNA duplexes,the receipient. The cut is enlarged to a gap by exonuclease action. The exonuclease nibbles away one strand on the either side of the break, generating a 3' single stranded terminii. One of the free 3' end than involves a homologus region in the other donor duplex. This is single strand invasion. The formation of heteroduplex DNA generates a D loop, in which one strand of the donor duplex is displaced. Eventually the D loop becomes large enough to correspond the entire length of the gap on the receipient chromatid. When the extruded single strand reaches the far side of the gap, the complementary single stranded sequences anneal. The structure contains a heteroduplex DNA on either side of the gap and the gap itself is represented by the single stranded D loop.
Branch migration converts this structure into a molecule with two recombinant joints. the joint must be resolved by cutting. If both the joints sre resolved in the same manner, the original non-crossover molecules will be released, each with aregion of altered genetic information. If the two joints are resolved in opposite ways, a genetic crossover is obtained.
Proteins involved in Recombinations:
There are various important proteins involved in homologus recombination. These are as follows:
Rec A:
The general recombination is catalyzed by Rec A protein. The E.coli protein Rec A was the first example to be discovered of DNA strand transfer protein. It is a paradigm for a group that includes several other bacteria and archael proteins. The Rec A protein is extremely significant to recombination and is indicated by two independent observations:
a) rec A- E.coli have a rate of recombination of 104 - fold lower than the wild type of E.coli
b) Rec A greatly increases the rate at which complementary strand renatures in vitro.
Rec A protein has 352 amino acid residues. For such a small protein it has too many activities. It can stimulate protease activity in the SOS response and can promote base pairing between single stranded DNA and its components in duplex molecule. It is a DNA binding protein which catalyzes strand exchange at the cost of ATP hydrolysis. The DNA binding proceeds from 5' end to 3' end. Rec A binds to single stranded DNA or even duplex DNA provided it has single stranded gap. the binding is cooperative and soon a filament of Rec A monomer gets created. This filament is a right handed helix with a pitch of of 95A° and ˜ 6.2 Rec A monomers per turn of the helix.
X-ray structure reveals that it is 120 A° wide and has a large helical groove which is about 25 A° wide. It is in this groove that DNA can be enclosed.
A remarkable feature of RecA - DNA binding is that Rec A engagges the minor groove of DNA. This ability of Rec A is important for strand exchange during recombination. Such a binding of Rec A leaves major groove accessible for a possible reaction with a second molecule of DNA. The DNA handling activity of Rec A enables a single strand to displace its homolog in a duplex in a reaction called assimilation.
Mechanism of Rec A:
Rec A binds to single stranded DNA. When a linear single strand invades a duplex, it displaces the original partner to its complement. This requires concurrent migration of Rec A nucleoprotein filament along the molecule. this can happen only in 5' → 3' direction. Partial unwinding of the DNA takes place.
In a rection needing ATP hydrolysis, Rec A exchanges its bound single strand with the homologus strand of the duplex. The presence of single strand binding protein stimulates the raction, by ensuring that the substrate lack secondary structure. Rec A is required in stoichoimetric amounts, which suggests that its action in strand assimilation involves binding cooperatively to DNA to form structure related to filaments.
RecBCD protein:
In bacterial conjugation and in phage transduction, a protein encoded by the genes recB, recC and recD is needed in addition to recA. These three genes code for three subunits of RecBCD protein. The RecBCD os a multifunctional protein. it has differen activities. The exonuclease activity was detected first and hence it is also called as exonucleaseV. It degrades DNA and has helicase activity that can unwind duplex DNA in presence of single strand binding protein. It also has ATPase activity.
About every 10kb in e.coli genome, there occurs a GCTGGTGG sequence, which is called as chi sequence. This chi sites are the targets for action of an enzyme encoded by RecBCD protein. When RecBCD binds DNA on the right side of chi, it moves along unwinding the DNA. While moving, it remains in contact with two regions of that strand, allowing the end of the strand to pass through the enzyme, thus forming a single strand loop. When it reaches the chisite, it pauses and cleaves one strand of the DNA at a position between 4 and 6 bases of 3' side of chi.
As the length of single strand tail increases, RecA binds to the sinfle strand and homologus base pairing occurs with the other strand to form the doubly looped structure. Recognition of the chi site causes RecD subunit to dissociate and becomes inactivate due to which it looses its nuclease activity, but it continues to function as helicase.
RUV proteins:
One of the most critical steps in recombination is the resolution of Holiday junction. The proteins involved in stabilizing and resolving Holiday junctions have been identified as the products of RUV genes in E.coli. These genes are ruvA, ruvB and ruvC and their protein products are RuvA, RuvB, RuvC respectively.
RuvA:
RuvA is atetramer. It is asmall protein whose function is to recognize the structure of Holiday junction. It binds to all four strands of DNA at the crossover point and forms two tetramers that sandwitch DNA.
RuvB:
RuvB is a hexameric helicase with an ATPase activity that provides the motor for branch migration. hexameric rings of RuvB bind around each duplex of DNA upstream of the crosover point. It cannot act alone because it cannot bind to DNA effeciently. RuvA binds specifically to the crossover point and assist RuvB binding. Thus RuvA and RuvB functions jointly and increases the heteroduplex formation together.
RuvAB:
RuvAB complex can cause the branch to migrate fast as 10-20bp/sec. It displaces RecA from DNA during its action. It can act of Holiday junction, but if it is mutant thaen it is completely defective in recombination activity.
RuvC:
RuvC codes for an endonuclease that specifically recognizes Holiday junction. It functions as a dimer to cleave two of the four strands that make up the central part of the recombination intermediate. A common tetranucleotide sequence provides hotspot for RuvC to resolve Holiday junctions. The tetranucleotide is asymetric and thus direct resolution with regard to which pair of strands to be nicked. This determines whether the outcome is patch recombinant or splice recombinant.
Site Specific Recombination :
It is a specialized type of recombination. Specialized recombination involves a reaction between two specific sites. The target sites are short. Sometimes the two sites have same sequence while sometimes they are homologous. The site specific reaction is used to insert a free phage DNA into the bacterial chromosome or to excise an integrated phage DNA from the chromosome. It is also used before division to regenerate monomeric circular DNA from a dimmer that has been created by generalized recombination event.
The enzymes that catalyze site-specific recombination are called as recombinase and those involved in phage integration are called as integrase. The prominent members of the integrase family are prototype ‘int’ from phage lambda, ‘cre’ from phage P1 and the yeast ‘FLP’ enzyme.
The classic model of site specific recombination is illustrated by phage lambda. The phage spends its life in a bacterium in two different styles:
a) Lytic phase:
In this phase, DNA is independent and can remain either circular, replicate itself several times over, getting assembled into the phage particle and finally lysing the host cell to come out and begin a fresh cycle.
b) Lysogenic cycle:
In this phase, the phage DNA can integrate into the host genome and replicate every time the host DNA does and thus spread in all the host progeny of the infected cell. This phage is called as prophage.
Transition between these two stages requires site specific recombination. To enter lysogeny, the phage must integrate itself in the DNA of host and to get back into lytic cycle, the prophage DNA must excise itself from the host DNA and released from chromosome.
Integration and excision occur by recombination at specific loci on the bacterial and phage DNA’s called attachment (att) sites. The attachment site on the bacterial chromosome is called as att in bacterial genetics. For describing the int/excision reaction, the bacterial attachment attachment sites are called as attB and consist of the sequence components BOB’. The attachment site on the phage, attP, consists of components POP’. The sequence ‘O’ is common to attB and attP. It is called as core sequence and the recombination events occur within it. The att sites have distinct requirements; attP is much larger than attB. The function of attP requires a stretch of 240bp while attB function can be excised at 23bp fragment. in which there are only 4bp on the either side of the core. The disparity in their sizes suggests that they play different roles with attP providing additional information necessary to distinguish it from attB.
The flanking regions B, B’ and P, P’ are called as arms, each is distinct in sequence. Because the phage DNA is circular, the recombination event inserts it into the bacterial chromosome as a linear sequence. There are new sites formed in the linear phage i.e. attL and attR. These two sites are the product of recombination. Integration requires the product phage gene int, which codes for an integrase enzyme, and a bacterial protein called as integration host factor (IHF). Excision requires the product of phage gene xis in addition to Int and IHF.
The site-specific recombination has been halted at intermediate stages by the use of suicidal substrates, in which the core sequence is nicked. The presence of nick interferes with the recombination process. This makes it possible to identify molecules in which the recombination has commenced, but has not been completed. The structure of these intermediates suggests that exchanges of single strand take place sequentially.
The steps of recombination events are:
1) The corresponding strands are cut on the phage and bacterial duplex. The cuts
are made at the same position.
2) The free 3’ends exchange between the two duplexes.
3) The branch migrates for a distance of 7bp along the region of homology.
4) The Holiday structure is resolved by cutting the other pair of corresponding strands.
Components of site specific recombination:
λ recombination occurs in large structure called intasome. It has different components for each direction of reaction:
1) IHF:
A host protein IHF is required for both integration and excision. It is 20 KD proteins of two different subunits coded by genes himA and himD. It is not an essential protein in E.coli. And is not required for bacterial recombination. It is one of the several proteins with the ability to wrap DNA on a surface.
Mutations in him genes prevent λ site specific recombination and can me prevented by mutation in λint. IHF binds to sequence of approximately 20bp in attP. The IHF binding sites are adjacent to int sites.
2) Int:
The int has two different modes of binding. The C-terminal domain behaves like the cre-recombinase. It binds to the inverted sites at the core sequence, positioning itself to make the cleavage and ligation reactions on each strand. The N-terminal domain binds to sites in arms of attP that have a different consensus sequence. This binding is responsible for the aggregation of subunits into the intasome. When int and IHF bind to attP, they generate a complex in which all binding sites are pulled together on the surface of protein. This complex is called intasome. For intasome formation, supercoiling of attP is required.
The attB has two int sites in its core. But int does not bind directly to attB in the form of free DNA. The intasome is the intermediate that captures attB. The initial recognition between attP and attB does not depend directly on DNA homology, but instead is determined by the ability of int proteins to recognize both att sequences. The two att sites are then brought together in an orientation predetermined by the structure on intasome.
3) Xis:
Xis are proteins that control direction. Its presence inhibits integration and promotes excision. It binds to attP at two different sites. Binding of xis changes the organization of DNA, so that it becomes inert as a substrate for integration enzyme. Int can form complex with attR only if xis is bound. Xis protein decides whether int will function for integration or excision.
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