recombination

Contents:

• Definition
• Examples
-In prokaryotic system
-In eukaryotic system
• Types
• Meselson and Wiegle exp.
• Holliday model
• Potter and Dressler’s evidence
• Meselson and Raddding
• Enzymology
• Conclusion


Recombination
Definition- Recombination is a process or set of processes by which DNA molecules interact with one another to bring about a rearrangement of the genetic information or content in an organism.

Examples of recombination in prokaryotic systems are:• Integration of the bacteriophage lambda prophage,
• Recombination of bacterial DNA following conjugation between bacteria,
• Formation of plasmid multimers.
Examples of recombination in prokaryotic systems are:• Meiosis crossing over- also called gemetic recombination.
• At the time of expression of antibody genes so as to generate great potential diversity of antibody molecules.

Types of recombination- 4 general types of recombination:
Homologous genetic recombinationAlso known as general recombination or general homologous recombination involves an exchange of genetic material between homologous chromosomes, resulting in the arrangement of genes into new combinations.
It is also called general recombination because the enzymatic machinery that mediates the exchange can use essentially any pair of homologous DNA sequences as substrates.
This is the type of recombination that is required during meiotic crossing over, for recombination following bacterial conjugation, and during the formation of plasmid multimers
Site-specific recombination
As the name implies, this type of recombination involves the exchange of genetic material at very specific sites only. Examples include the integration of bacteriophage lambda into the host chromosome to form the prophage and the rearrangement of chromosomal DNA prior to expressing antibody genes.
DNA transpositionServes as mechanism by which particular DNA sequences called transposons may be moved from one location to another on chromosomes independently.
Illegitimate recombinationA very rare form of recombination, occurs between non homologous DNA independently of any unique sequence element.

Molecular mechanism of recombination-
The Meselson - Weigle Experiment
In the simplest sense, recombination is an exchange of both strands between two DNA molecules:

This representation implies that both strands of each molecule must be broken and then rejoined. This was first demonstrated by an experiment performed by Matt Meselson and Jean Weigle in 1961.
Meselson and Weigle infected E. coli cells at the same time with phage from two different stocks of bacteriophage lambda. One stock had been prepared by growing the bacteriophage lambda c-mi- in cells grown in medium containing heavy isotopes of carbon (13C) and nitrogen (15N). The other stock had been prepared by growing bacteriophage lambda c+mi+ in medium containing light isotopes of carbon and nitrogen.


After infection, the progeny phage were isolated and banded on a CsCl gradient.
A broad band of phage particles were found on the gradient.
• Nonrecombinant phage were found, as expected, at two well-defined densities corresponding to the parental light and heavy phages.
• Recombinant phage were found - surprisingly - at all intermediate densities between these two.



They also followed the course of the infection using two genetic markers, c and mi, which were located near one end of the lambda chromosome.

When the phenotypes of the intermediate density phage particles were analyzed, recombinant phage that were c-mi+ were found near the band of "heavy" phage while recombinant phage that were c+mi- were found near the band of "light" non-recombinant phage.
These results can only be explained if recombination between the two parental phage involves breakage and rejoining of both DNA strands (as shown above).

The Holliday Model of Genetic Recombination
This model of recombination was first proposed by Robin Holliday in 1964 and re-established by David Dressler and Huntington Potter in 1976 who demonstrated that the proposed physical intermediates existed.
The basic (simple) model
Align two homologous DNA molecules.

Nick the DNA at the same place on the two molecules.
This must happen in strands with the same polarity.

Exchange strands and ligate.The intermediate that is formed is called a Holliday intermediate or Holliday structure. The shape of this intermediate in vivo is similar to that of the greek letter chi, hence this is also called a chi form.

Visualization of the next step is made easier if one molecule is now rotated through 180š with respect to the other. This also helps to emphasize the chi-shape of the intermediate:


Resolve the structure.There are two ways in which this can happen:
• If the same strands are cleaved a second time then the original two DNA molecules are generated:

• If the other strands are cleaved, then recombinant molecules are generated:


A more realistic model
The above model is too simple and does not explain a number of genetic results, including the occurence of two different recombinant bacteriophage in a single plaque in the Meselson-Weigle experiment.
These can be explained by modifying the model slightly. As before, two homologous DNA molecules must be aligned and nicked at the same place. Following strand exchange the intermediate Holliday structure is formed. At this stage a new step is introduced:
Branch migration.
Migration of the branch can occur over many nucleotides in either direction. The result is a physical transfer of part of one of the strands of one molecule with that of the other:

Once again, visualization of the next step is made easier if one molecule is now rotated through 180š with respect to the other.

Resolve the structure.
There are still two ways in which this can happen, however, the consequences are different:

• If the same strands are cleaved a second time then nonrecombinant DNA molecules are generated but they each contain a region of heteroduplex DNA that spans the region of branch migration. Such heteroduplexes are called Patch recombinants.

• If the other strands are cleaved, then recombinant molecules are generated as before, however, each will also contain a region of heteroduplex DNA that spans the region of branch migration. Such heteroduplexes are called Splice recombinants.


Potter & Dressler's evidence for the Holliday ModelIn 1976, David Dressler and Hunt Potter published the results of a series of experiments that demonstrated the validity of the Holliday model of recombination.
They used E. coli cells containing the colicin E1 derived plasmid, pMB9. This plasmid was one of the very earliest plasmids developed for cloning in Herbert Boyer's laboratory. Normally, E. coli contain about 20 copies of this plasmid per cell. However, if the cells are exposed to chloramphenicol then, although chromosomal replication stops, plasmid replication does not and the number of plasmid molecules increases to 1000 copies per cell. With so many more copies of the plasmid in the cell, the chances of recombination increase as does the probability of observing a recombination intermediate.
When plasmid was isolated from the cells, purified by CsCl gradient centrifugation, and observed in the electron microscope, a number of candidates for intermediates were observed. These all had the appearance of "figure 8" structures. However, there are 3 possible ways such structures might arise:
• as a double-sized circular plasmid twisted over on itself.
• as two interlocking circular plasmid molecules.
• as a genuine recombination intermediate.
In order to distinguish between the three possibilities, Potter and Dressler digested their plasmid preparations with EcoRI. This enzyme will generate monomer sized linear molecules from either of the first two possible structures. However, it will generate unique chi-shaped structures from the third.
When they did this, Potter and Dressler found that between 0.5% and 3% of the molecules they observed were chi-shaped structures. The molecules were symmetrical in that the opposite arms were identical lengths and had identical denaturation patterns. Finally, they saw no such structures if they prepared their plasmids from recA- strains of E. coli.
From this evidence they concluded:
. . . the intermediates we have observed in the electron microscope provide physical evidence in support of the recombination intermediate postulated by Holliday on genetic grounds.


Matt Meselson And Charles Radding Models of Genetic RecombinationAlthough, the basic features of the Holliday model are well-established, the model does have flaws. For example, the mechanism by which two homologous regions of DNA are paired and then nicked is not well explained. In addition, the model does not explain all of the observed results in different recombination systems.
A central feature of the Holliday model as outlined above is that the heteroduplex regions in recombinant molecules will be identical in length and position. Experimental results suggest, however, that this is not necessarily so.



In order to explain such discrepancies, Matt Meselson and Charles Radding proposed a modified model. In their model, only one strand in one of the two paired homologous DNA molecules is nicked. This strand is then displaced by DNA polymerase I and invades the homologous chromosome. A Holliday intermediate is eventually formed and resolved as above. In this model, the extent of heteroduplex DNA in the resolved products, whether recombinant or nonrecombinant, will be different. Finally, gene conversion (which is a type of recombination event) in yeast follows a mechanism, proposed by Szostak, that requires double-strand breaks in one of the recombining molecules.

Enzymology of general recombination-Recombination Proteins in E. coli
The characterization of the structures and biochemical function of a number of the key proteins required for recombination is now helping us to understand details of the mechanism of recombination. The most important proteins are RecA, RecBCD, RuvA, RuvB and RuvC.

RecBCD The recB, recC & recD genes code for the three subunits of the RecBCD enzyme which has five activities: exonuclease V; a helicase activity; an endonuclease activity; an ATPase activity; and, an ssDNA exonuclease activity.
The RecBCD helicase activity can unwind DNA faster than it rewinds. Thus as it travels along a DNA molecule, it can generate ssDNA loops.
The RecBCD complex functions as a DNA exonuclease. It will bind to double-stranded breaks in DNA and degrade both strands simultaneously. However, when RecBCD encounters a Chi sequence, its activity changes. These Chi sites are recombinational Hot Spots. Characterized by 5’-GCTGGTGG-3’. Rec BCD cleaves DNA strand 4-6 nucleotides to 3’ side of Chi. The RecD subunit is released and the RecBC proteins act as a helicase to unwind the DNA in an ATP dependent reaction. This generates a ssDNA region that can serve (along with RecA) to initiate strand exchange and a recombination reaction.
the RecBCD pathway illustrates this:




RecAThe RecA protein is a multifunctional powerhouse! It has strand-exchange, ATPase and co-protease activities all packed into a compact 352 amino-acid, 38 kDa structure. It is required for all recombination pathways in E. coli.
The RecA protein will bind cooperatively to a ssDNA molecule with each monomer of RecA binding to a span of 4-6 nucleotides. Assembly of the nucleoprotein complex proceeds in a 5' -> 3' direction. The complex is both fairly stable (half-life is 30 min) and is the active species that will promote strand exchange.
The RecA protein monomer structure is shown above. Monomers associate with one another via the N-terminal alpha helix which interacts with the adjacent monomer -- forming a molecular conga line, if you will, with one helix on the shoulder of the molecule in front of it! The RecA filament that forms is helical with a pitch of 82.7 Å and it consists of 6 monomer units per turn.
RecA will promote strand exchange between DNA molecules as long as the following conditions apply:
• One of the two molecules must have a ssDNA region to which RecA can bind.
• The two molecules must share a region of homologous (i.e. nearly identical) DNA sequence - a minimum of 50 bp is required.
• There must be a free end within this region of homology which can initiate the strand exchange.
The strand exchange reaction probably involves the following steps:
• RecA binds to the ssDNA partner.
• The two molecules are aligned possible through the formation of a triple-stranded intermediate.
• Displacement of one of the old strands. This requires concurrent migration of the RecA nucleoprotein filament along the molecule - which proceeds in one direction only (5' -> 3') - and consequent winding/unwinding. ATP hydrolysis takes place during this step.
Proteins required for Resolving Holliday Junctions in E. coli

RuvARuvA is a small protein whose function is to recognize a Holliday junction thereby assisting the RuvB helicase to promote branch migration.
The crystal structure of the E. coli RuvA protein was solved at a resolution of 1.9 Å. The protein forms a tetramer in an unusual manner - though one that is ideally suited to its function. To quote from the summary part of the paper:
"Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction."
The RuvA protein is 203 amino acids in length but only 190 of them could be assigned in the crystal structure. Most of the missing assignments represent amino acids in a flexible part of the protein.





More recently, the crystal structure of RuvA complexed with DNA has been solved first at 6.0 A and then at 3.1 A.
Finally, examine these three model structures showing how a Holliday intermediate could interact with RuvA:






The RuvA-Holliday junction complexes have the predicted structure with a four-fold symmetry. The DNA bound at the junction is not flat but slightly concave to maximize the contact between the DNA and the protein and, as a result, the axes of the four helices are inclined at about 10 degrees. In the centre of the complex, eight residues corresponding to Glu-55 and Asp-56 from each of the four subunits, form an acidic central pin which may repel the DNA backbone away from the centre of the junction thereby, opening up the centre.
These refined structures have also shown that there are only 4 unpaired bases in the centre of the complex. This is a lot fewer than found for typical helicases.

RuvBThe RuvB protein is a helicase that catalyzes branch migration of Holliday junctions. By itself it cannot bind to DNA efficiently. It functions in combination with RuvA. Like other helicases, RuvB functions as a hexamer; but, unlike other helicases, RuvB encloses double-stranded DNA not ssDNA.
Electron microscopy has shown that RuvB is a heptamer in solution and that it converts to a hexamer ring when it binds to DNA. Electron microscopy has also shown that the two hexamer rings of RuvB lie contacting RuvA on the two opposite sides of a RuvAB-Holliday junction complex.
RuvCThe RuvC protein resolves the Holliday intermediate. It functions as a dimer to cleave two of the four strands that make up the central part of the intermediate. Since binding is symmetrical, RuvC can bind to the Holliday intermediate in two equally likely ways. Hence, Holliday intermediates can be resolved in two different, but equally likely, ways.
RuvC does have some sequence specificity. It cleaves DNA at the 3'-side of thymidine, preferentially at the consensus 5'-A/TTT|C/G -3' where | indicates the site of cleavage
The interaction of RuvC with Holliday junction is shown:


A Model for Resolving Holliday Junctions in E. coliThe crystal structures of RuvA and RuvC, as well as detailed electron microscope visualization of Holliday junction protein complexes allow for a reasonable model to be drawn showing how branch migration occurs and how the junction is resolved:

It i sknown now that RuvC functions with RuvA and RuvB in a higher order complex, termed the RuvABC resolvasome. However, it is also clear that the known structure of RuvC cannot be docked with the detailed RuvA-Holliday complex structure - steric conflicts are found. It is believed that there must be some other structural changes that occur within the resolvasome; but, these have yet to be identified.
The structure of RuvB at 1.9A has been determined recently. The proposed model of the complete resolvasome fits well with the hypothetical models have have been devised based on the structures of RuvA and RuvC. It has also been possible to devise a model to explain DNA translocation through RuvB as a result of ATP hydrolysis.

Conclusion-
• Chromosomal segregation, shuffles together the genetic material carried by different members of a sexual species.
• This genetic mixing unties the evolutionary fate of alles at one locus from the fate of alleles at neighboring loci and can increase the amount of genetic variation found within a population.
• In the process, however, recombination separates advantageous gene combinations, the very gene combinations that enabled the parents to survive and reproduce.
• Whether or not the adaptation of a population to an environment is more rapid in the presence of recombination, that is, whether or not recombination speeds up the evolutionary process, depends critically on the ways in which this process is modelled. As we shall see, the effect of recombination depends on the population size, the initial population composition, and the selection regime under consideration.