2. Breakage and reunion involves heteroduplex DNA

14.2 Breakage and reunion involves heteroduplex DNA

Key terms defined in this section
Branch migration describes the ability of a DNA strand partially paired with its complement in a duplex to extend its pairing by displacing the resident strand with which it is homologous.
Hybrid DNA is another term for heteroduplex DNA.
Recombinant joint is the point at which two recombining molecules of duplex DNA are connected (the edge of the heteroduplex region).



Figure 14.1 Recombination occurs during the first meiotic prophase. The stages of prophase are defined by the appearance of the chromosomes, each of which consists of two replicas (sister chromatids), although the duplicated state becomes visible only at the end. The molecular interactions of any individual crossing-over event involve two of the four duplex DNAs.

The act of connecting two duplex molecules of DNA is at the heart of the recombination process. Our molecular analysis of recombination therefore starts by expanding the view in Figure 14.1 of the use of base pairing between complementary single strands in recombination. It is useful to imagine the recombination reaction in terms of single-strand exchanges (although we shall see that this is not necessarily how it is actually initiated), because the properties of the molecules created in this way are central to understanding the processes involved in recombination.




Figure 14.2 Recombination between two paired duplex DNAs could involve reciprocal single-strand exchange, branch migration, and nicking.

Figure 14.2 illustrates a process that starts with breakage at the corresponding points of the homologous strands of two paired DNA duplexes. The breakage allows movement of the free ends created by the nicks. Each strand leaves its partner and crosses over to pair with its complement in the other duplex.


The reciprocal exchange creates a connection between the two DNA duplexes. The connected pair of duplexes is called a joint molecule. The point at which an individual strand of DNA crosses from one duplex to the other is called the recombinant joint.


At the site of recombination, each duplex has a region consisting of one strand from each of the parental DNA molecules. This region is called hybrid DNA or heteroduplex DNA.




Figure 14.3 Branch migration can occur in either direction when an unpaired single strand displaces a paired strand.

An important feature of a recombinant joint is its ability to move along the duplex. Such mobility is called branch migration. Figure 14.3 illustrates the migration of a single strand in a duplex. The branching point can migrate in either direction as one strand is displaced by the other.


Branch migration is important for both theoretical and practical reasons. As a matter of principle, it confers a dynamic property on recombining structures. As a practical feature, its existence means that the point of branching cannot be established by examining a molecule in vitro (because the branch may have migrated since the molecule was isolated).


Branch migration could allow the point of crossover in the recombination intermediate to move in either direction. The rate of branch migration is uncertain, but as seen in vitro is probably inadequate to support the formation of extensive regions of heteroduplex DNA in natural conditions. Any extensive branch migration in vivo must therefore be catalyzed by a recombination enzyme.




Figure 14.4 Resolution of a Holliday junction can generate parental or recombinant duplexes, depending on which strands are nicked. Both types of product have a region of heteroduplex DNA.

When recombination involves duplex DNA molecules, topological manipulation may be required; either the DNA duplex must be free to rotate, or equivalent relief from topological restraint must be provided (see later). If we imagine that the joint molecule of Figure 14.2 rotates one duplex relative to the other, we can visualize it in one plane as a Holliday structure (named for its proposer). This is illustrated in Figure 14.4.


The joint molecule formed by strand exchange must be resolved into two separate duplex molecules. Resolution requires a further pair of nicks. The outcome of the reaction depends on which pair of strands is nicked, as can be seen from Figure 14.2 and Figure 14.4.


If the nicks are made in the pair of strands that were not originally nicked (the pair that did not initiate the strand exchange), all four of the original strands have been nicked. This releases splice recombinant DNA molecules. The duplex of one DNA parent is covalently linked to the duplex of the other DNA parent, via a stretch of heteroduplex DNA. There has been a conventional recombination event between markers located on either side of the 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 each has a residuum of the event in the form of a length of heteroduplex DNA. These are called patch recombinants.


These alternative resolutions of the joint molecule 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 recombination of the flanking regions.


What is the minimum length of the region required to establish the connection between the recombining duplexes? Experiments in which short homologous sequences carried by plasmids or phages are introduced into bacteria suggest that the rate of recombination is substantially reduced if the homologous region is <75 bp. This distance is appreciably longer than the ~10 bp required for association between complementary single-stranded regions, which suggests that recombination imposes demands beyond mere annealing of complements.




Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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