Of Each Molecule

Migration

The mechanism of crossover formation involves a temporary triple helix and specific recognition sequences, the chi sites.

FIGURE 14.04 Rearrangement and Resolution of a Holliday Junction

The Holliday junction can isomerize or change into two alternate conformations. Resolution of form I exchanges gene "B" from the pink molecule with gene "b" from the purple molecule resulting in a patch recombinant. The other strands of the two DNA molecules are unaffected, thus only a small region of heteroduplex DNA results. When form I isomerizes, an intermediate "chi form" appears first, then the crossover reforms. Notice that form I and II have identical base pairing. The difference between forms I and II is the crossover arrangement. In form I, the broken and rejoined strands crossover whereas in form II, the unbroken strands crossover. Resolution by RuvC hybridizes both DNA strands of the double helix rather than just one.

FIGURE 14.05 Migration of a Holliday Junction

A complex of four RuvA proteins and six RuvB is able to break and reform hydrogen bonds between base pairs thus allowing the crossover to migrate along the DNA helix.

FIGURE 14.05 Migration of a Holliday Junction

A complex of four RuvA proteins and six RuvB is able to break and reform hydrogen bonds between base pairs thus allowing the crossover to migrate along the DNA helix.

contacts all 4 strands

then intrudes into the second DNA double helix to give a triple-stranded helix. The two stages in this process depend on the RecBCD and RecA proteins, respectively. Originally it was thought that crossovers could form between any two homologous sequences, but specific sequences called chi sites are also needed. However, since chi sequences (5'-GCTGGTGG-3') are very common, crossovers do form more or less at random in any sufficient length of bacterial DNA. The chi site was named because of the resemblance of a crossover to the Greek letter chi, %.

During single-stranded invasion, RecBCD binds to DNA at double-strand breaks. It then moves along the DNA unwinding the double helix until it reaches a chi site (Fig. 14.06). Here the RecD endonuclease clips one of the strands to the 3'-side of the chi sequence and then dissociates from the DNA. The RecBC helicase continues unwinding the DNA generating a single strand.

The RecA protein binds to the single strand with the free 3'-end and inserts it into another DNA double helix to give a temporary triple helix (Fig. 14.07). RecA stabilizes the single-stranded region of DNA. This strand invasion causes displacement of one of the strands of the second double-helix. This displaced strand will eventually pair with the remaining single strand of the DNA that was originally unwound by RecBCD. The resulting crossover is resolved as described in Fig. 14.04, above.

The question remains—how did the double-stranded break appear in the first place? Bacteria avoid generating double-stranded breaks in their chromosomes. Furthermore, bacteria are haploid, therefore, do not contain pairs of homologous chromosomes that recombine during sexual reproduction. In practice, recombination in bacteria occurs between the resident bacterial chromosome and shorter fragments of incoming DNA. These fragments enter the bacterial cell by a variety of processes (see Ch. 18 for details). They may be taken up as free DNA from the outside medium (transformation), carried inside a virus particle (transduction), or received from another cell during mating (conjugation). In most cases the incoming DNA will consist of relatively short linear fragments that provide the ends necessary for recognition by RecBCD. These mechanisms provide genetic diversity to haploid, non-sexual bacteria, allowing them to adapt to changing environments.

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