So far we have talked a lot about recombination: estimation of recombination frequencies and construction of genetic maps based on linkage relationships derived from recombination frequency data. We now want to look at the mechanism(s) of recombination: how does recombination take place and what is the molecular basis of recombination.
 

Generalized recombination
 

This is the mechanism that mediates most forms of chromosomal exchange. General recombination occurs between homologous DNA molecules or homologous chromatids at meiosis and involves breakage and reunion of these homologous DNA molecules. How this is achieved is not absolutely certain and a number of models have been proposed to explain the processes involved.
 
 

The Holliday model
 
 

Proposed in 1964 by Robin Holliday, this has subsequently refined by a number of geneticists, particularly Meselson & Radding (1975) and Holliday (1974) to give what is sometimes called the Meselson & Radding model, but is better described as a modified Holliday model.
 
 
 
 

The first step is the pairing of DNA duplexes, i.e. homologous DNA regions recognise each other and become aligned. How this is achieved is not understood, though in eukaryotes this involves the formation of synaptonemal complexes that mediate the meiotic exchange of genetic information. By some mechanism, the base pairs recognise each other in different duplexes so that homologous regions become aligned.
 
 

Fig 1: Two DNA molecules become aligned
 
 

The next stage is the formation of nicks in homologous strands. This is done by endonucleolytic cleavage.
 
 

Fig 2: Formation of nicks
 
 

The breakage allows movement of the free ends created by the nicks. Each strand leaves its partner and crosses over to pair with its complementary strand in the other duplex.
 
 

This reciprocal exchange creates a connection between the two DNA duplexes. Initially this is sustained only by H-bonding, but at some stage covalent bonds are formed by sealing the nicks at the site of exchange with DNA ligase. The connected pair of duplexes is called a joint molecule or a Holliday intermediate.
 
 

Fig 3a, b, c: crossing-over and ligation
 
 
 
 

At the site of recombination, each duplex has a region consisting of one strand from each region of the parental DNA molecules. This region is called hybrid or heteroduplex DNA. The position where the two strands swap over each other is known as the branch and once the strand exchange has been initiated it can move along the duplex - this is known as branch migration.
 
 

Fig 4: Branch migration
 
 

The rate of branch migration appears to be fast enough (> 1000 bp/sec) to support the formation of extensive regions of heteroduplex DNA in natural conditions. To achieve this, topological manipulation may be required to rotate the DNA and this is achieved by a topoisomerase enzyme.
 
 

Also, by rotational means, the Holliday intermediate can generate different planar configurations.
 
 

Fig 5: Holliday intermediates
 
 

These planar intermediates have the shape of the Greek letter chi (c) and are referred to as the chi-form. These chi-form intermediates have been visualised in certain situations by electron microscopy (Fig 13.32. PrOG).
 
 

The joint molecule formed by strand exchange must be resolved back into two separate duplex molecules. This requires a further pair of endonuclease nicks and these can occur in two orientations: vertical or horizontal.
 
 

Fig 6: Cleavage of Holliday intermediates
 
 

This gives rise to recombination of the flanking markers, i.e. two spliced reciprocal recombinant duplexes. This does not give rise to recombination if the flanking markers but gives rise to two patched duplexes with central sectors of heteroduplex DNA.
 
 

The Meselson and Radding variation
 
 

This is the basic model, but there are slight variations. For example, as Meselson and Radding (1975) proposed, the initial strand exchange would involve only one strand instead of both - leading to formation of a D-loop. Whether the situation is one or two strand initiation has not been resolved, but all variations of the model rely on the same general principle: the formation of an intermediate involving heteroduplex DNA that can be extended by branch migration.
 
 

The Szostak variation
 
 

A more recent variation of the model is one proposed by Szostak and co-workers (1983) and this introduces the idea of the double strand break. Recombination is initiated when an endonuclease makes a double strand break in one chromatid (the Arecipient@) (Fig 7a). This break is enlarged to a gap, probably by exonuclease action which generates 3' single-stranded termini.
 
 

Fig 7a, b: Cleavage and digestion
 
 

One of the free ends then invades a homologous region in the other adjacent (Adonor@) duplex and the formation of heteroduplex DNA generates a D-loop. The D-loop is then extended by a repair synthesis that uses the free 3' end as a primer to which a newly synthesized DNA strand can be added.

Fig 8: D-loop formation and extension
 
 

The duplex integrity of the gapped recipient can be achieved by a second round of single-stranded DNA synthesis using the left-hand-side 3' end as a primer. Branch formation in this region can lead to formation of a molecule with two recombinant joints.
 
 

This molecule can be resolved back to two DNA molecules by strand cleavage. If both junctions are resolved in the same way e.g. the inner strands are cut at each joint then non-cross over molecules will be released. Each will have an altered region of genetic information that is a footprint of the exchange event.
 
 

Fig 9a, b, c:
 
 
 
 

If the two junctions are resolved in opposite ways, then a genetic crossover will result, i.e. one is cut on the inner strand and the other on the other.
 
 

Fig 9d
 
 

In this model, then, two regions of heteroduplex DNA are formed. One at each end of the region involved in the exchange. And this region is in the middle which corresponds to the original to the original gap in the recipient DNA molecule now has the sequence of the donor DNA in both molecules.
 
 

So the arrangement of the heteroduplex sequences is asymmetric and part of one molecule has been converted to the sequence of the other (which is why the initiating chromatid is called the recipient).
 
 

Data obtained with yeast are consistent with the double-strand break repair molecule, i.e. that initiation is involved with receiving genetic information:
 
 

3) Many yeast and Drosophila mutants are hypersensitive to particular mutagens (repair defective) and these are often also defective in the recombinational steps of meiosis.

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