6. RecA catalyzes single-strand assimilation

14.6 RecA catalyzes single-strand assimilation

Key terms defined in this section
Paranemic joint describes a region in which two complementary sequences of DNA are associated side by side instead of being intertwined in a double helical structure.
Single-strand assimilation describes the ability of RecA protein to cause a single strand of DNA to displace its homologous strand in a duplex; that is, the single strand is assimilated into the duplex.

RecBCD-mediated unwinding and cleavage can be used to generate ends that initiate the formation of heteroduplex joints. The enzyme RecA can take the single strand with the 3′ end that is released when RecBCD cuts at chi, and can use it to react with a homologous duplex sequence, thus creating a joint molecule.


RecA has two quite different types of activity: it can stimulate protease activity in the SOS response (see later); and can promote base pairing between a single strand of DNA and its complement in a duplex molecule.


RecA requires single-stranded DNA and ATP for ability to stimulate protease activity. The same substrates are required for its ability to manipulate DNA molecules. It is not yet clear exactly how the enzymatic activities of RecA are related to recombination in vivo, but they involve several reactions that provide useful paradigms for recombination mechanisms.


The DNA handling activity of RecA enables a single strand to displace its homolog in a duplex in a reaction that is called single-strand uptake or single-strand assimilation. The displacement reaction can occur between DNA molecules in several configurations and has three general conditions:



  • One of the DNA molecules must have a single-stranded region.
  • One of the molecules must have a free 3′ end.
  • The single-stranded region and the 3′ end must be located within a region that is complementary between the molecules.



Figure 14.10 RecA promotes the assimilation of invading single strands into duplex DNA so long as one of the reacting strands has a free end.

The reaction is illustrated in Figure 14.10. When a linear single strand invades a duplex, it displaces the original partner to its complement. The reaction can be followed most easily by making either the donor or recipient a circular molecule. The reaction proceeds 5′ V3′ along the strand whose partner is being displaced and replaced, that is, the reaction involves an exchange in which (at least) one of the exchanging strands has a free 3′ end.




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


Figure 14.5 Recombination is initiated by a double-strand break, followed by formation of single-stranded 3 F ends, one of which migrates to a homologous duplex.

Single-strand assimilation is potentially related to the initiation of recombination. All models call for an intermediate in which one or both single strands cross over from one duplex to the other (see Figure 14.2 and Figure 14.5). RecA could catalyze this stage of the reaction.


A mechanism for the activity of RecA in stimulating branch migration is suggested by its ability to aggregate into long filaments with single-stranded or duplex DNA. There are 6 RecA monomers per turn of the filament, which has a helical structure with a deep groove that contains the DNA. The stoichiometry of binding is 3 nucleotides (or base pairs) per RecA monomer. The DNA is held in a form that is extended 1.5 times relative to duplex B DNA, making a turn every 18.6 nucleotides (or base pairs). When duplex DNA is bound, it contacts RecA via its minor groove, leaving the major groove accessible for possible reaction with a second DNA molecule.


The interaction between two DNA molecules occurs within these filaments. When a single strand is assimilated into a duplex, the first step is for RecA to bind the single strand into a filament. Then the duplex is incorporated, probably forming some sort of triple-stranded structure. In this system, synapsis precedes physical exchange of material, because the pairing reaction can take place even in the absence of free ends, when strand exchange is impossible.


A free 3′ end is required for strand exchange. The reaction occurs within the filament, and RecA remains bound to the strand that was originally single, so that at the end of the reaction RecA is bound to the duplex molecule. Large amounts of ATP are hydrolyzed during the reaction. The ATP may act through an allosteric effect on RecA conformation. When bound to ATP, the DNA-binding site of RecA has a high affinity for DNA; this is needed to bind DNA and for the pairing reaction. Hydrolysis of ATP converts the binding site to low affinity, which is needed to release the heteroduplex DNA.


We can divide the reaction that RecA catalyzes between single-stranded and duplex DNA into three phases:



  • a slow presynaptic phase in which RecA polymerizes on single-stranded DNA;
  • a fast pairing reaction between the single-stranded DNA and its complement in the duplex to produce a heteroduplex joint;
  • a slow displacement of one strand from the duplex to produce a long region of heteroduplex DNA.

The presence of SSB (single-strand binding protein) stimulates the reaction, by ensuring that the substrate lacks secondary structure. It is not clear yet how SSB and RecA both can act on the same stretch of DNA. Like SSB, RecA is required in stoichiometric amounts, which suggests that its action in strand assimilation involves binding cooperatively to DNA to form a structure related to the filament.


When a single-stranded molecule reacts with a duplex DNA, the duplex molecule becomes unwound in the region of the recombinant joint. The initial region of heteroduplex DNA may not even lie in the conventional double helical form, but could consist of the two strands associated side by side. A region of this type is called a paranemic joint (compared with the classical intertwined plectonemic relationship of strands in a double helix). A paranemic joint is unstable; further progress of the reaction requires its conversion to the double Vhelical form. This reaction is equivalent to removing negative supercoils and may require an enzyme that solves the unwinding/rewinding problem by making transient breaks that allow the strands to rotate about each other (see later).


All of the reactions we have discussed so far represent only a part of the potential recombination event: the invasion of one duplex by a single strand. Two duplex molecules can interact with each other under the sponsorship of RecA, provided that one of them has a single-stranded region of at least 50 bases. The single-stranded region can take the form of a tail on a linear molecule or of a gap in a circular molecule.




Figure 14.11 RecA-mediated strand exchange between partially duplex and entirely duplex DNA generates a joint molecule with the same structure as a recombination intermediate.

The reaction between a partially duplex molecule and an entirely duplex molecule leads to the exchange of strands. An example is illustrated in Figure 14.11. Assimilation starts at one end of the linear molecule, where the invading single strand displaces its homolog in the duplex in the customary way. But when the reaction reaches the region that is duplex in both molecules, the invading strand unpairs from its partner, which then pairs with the other displaced strand.




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.

At this stage, the molecule has a structure indistinguishable from the recombinant joint in Figure 14.4. The reaction sponsored in vitro by RecA can generate Holliday junctions, which suggests that the enzyme can mediate reciprocal strand transfer. We know less about the geometry of four Vstrand intermediates bound by RecA, but presumably two duplex molecules can lie side by side in a way consistent with the requirements of the exchange reaction.




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

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