F

base-pairing between strands is switched

Once a region of base-pair eomplimentarity is located. RecA promotes the formation of a stable complex between these two DNA molecules. This RecA-bound three-stranded structure is called a joint molecule and usually contains several hundred base pairs of hybrid DNA. It is within this joint molecule that die actual exchange of DNA strands occurs. The DNA strand in the primary binding site becomes base-paired with its complement in the DNA duplex bound in the secondary site. Strand exchange thus requires the breaking of one set of base pairs and the formation of a new set of identical base pairs. Completion of strand exchange also requires that the two newly-paired strands be intertwined to form a proper double helix. RecA binds preferentially to the DNA products after strand exchange has occurred and it is this binding energy that actually drives the exchange reaction toward the new DNA configuration.

RecA Homologs Are Present in All Organisms

Strand-exchange proteins of the RecA family are present in all forms of life. The best-characterized members are RecA from Fubacteria, RadA

fronl Archaea, RadSl and Dmcl from Fukaryota, and the bacteriophage T4 UvsX protein. These proteins form similar filaments to that made by RecA (Figure 10-11) and likely function in an analogous manner (although some features of the proteins are specifically tailored Tor their specific cellular roles and interaction partners). We will discuss the roles of Rad51 and Dmc1 recombination in eukaryotic cells below.

RuvAB Complex Specifically Recognizes Ho Hi day Junctions and Promotes Branch Migration

After the strand invasion step of recombination is complete, the two re-combining DNA molecules are connected by a DNA branch known as a Holliday junction (see above). Movement of the site of this branch requires exchange of DMA base pairs between the two homologous DMA duplexes. Cells encode proteins that greatly stimulate the rate of branch migration.

RuvA protein is a Holliday junction specific DNA-binding protein that recognizes the structure of the DMA junction, regardless of its specific DNA sequence. RuvA recognizes and binds to Holliday junctions and recruits the RuvB protein to this site. RuvB is a hexameric ATPase, similar to the hexameric helicases involved in DNA replication (see Chapter 8). The RuvB ATPase provides the energy to drive the exchange of base pairs that move the DNA branch. Structural models for RuvAB complexes at a Holliday junction show how a tetramer of RuvA, together with two hexamers of RuvB work together to power this DNA exchange process (Figure 10-12).

RuvC Cleaves Specific DNA Strands at the Holliday Junction to Finish Recombination

Completion of recombination requires that the Holliday junction (or junctions) between the two recombming DNA molecules be resolved, hi bacteria, the major Holliday junction resolving endonuclease is RuvC. RnvC was discovered and purified based on its ability tn cut

FIGURE 10-II RecA-lifceproteinsfn three branches of life. Nudeoprotein filaments are shown for (a) human fiac&l, (b) E coli RecA, and (c) A fulgidus RadA proteins, the Radii! and RecA proteins are also shewn in figuse ]0 8. Notice the similar helical structure of the filaments revealed by the stapes in these EM images. {Source: West S.C. et ai. Nature Reviews in Molccular and Cell Biology 4: 1-12. Images provided by A. Stasia^ University of Lausanne, Switzerland.)

FIGURE 10-II RecA-lifceproteinsfn three branches of life. Nudeoprotein filaments are shown for (a) human fiac&l, (b) E coli RecA, and (c) A fulgidus RadA proteins, the Radii! and RecA proteins are also shewn in figuse ]0 8. Notice the similar helical structure of the filaments revealed by the stapes in these EM images. {Source: West S.C. et ai. Nature Reviews in Molccular and Cell Biology 4: 1-12. Images provided by A. Stasia^ University of Lausanne, Switzerland.)

FIGURE 10-12 High resolution structure of RuvA and schematic model of the RuvAB complex bound to Holliday junction DNA, (a) The crystal structure of the RuvA tetramer shows the fourfold symmetry of the protein. (Ariyoshi M, Nishino T, Iwasaki H, Shmagawa H, and Morrkawa K. 2000. Proc Natl. Acad So. USA 97 8257-8262.) Image prepared with BobScript, MolScript, and Raster ¿D (b) A schematic model of the crystal structure is shown with two RuvB hexamers. Notice how a tetramer of RuvA hinds with fourfold symmetry to the junction. Two hexatners of RuvE hind a' opposite sfdes of RuvA arid function as a motor to pump DMA through the junction. The RuvB hcxamers are shown in cross-sections, so that the DNA threading through these complexes can be seen. (Source. From Varnada IC et at. Crystal structure of the RuvA RuvB complex. Mol Cell 10 677, fig. 4.)

FIGURE 10-12 High resolution structure of RuvA and schematic model of the RuvAB complex bound to Holliday junction DNA, (a) The crystal structure of the RuvA tetramer shows the fourfold symmetry of the protein. (Ariyoshi M, Nishino T, Iwasaki H, Shmagawa H, and Morrkawa K. 2000. Proc Natl. Acad So. USA 97 8257-8262.) Image prepared with BobScript, MolScript, and Raster ¿D (b) A schematic model of the crystal structure is shown with two RuvB hexamers. Notice how a tetramer of RuvA hinds with fourfold symmetry to the junction. Two hexatners of RuvE hind a' opposite sfdes of RuvA arid function as a motor to pump DMA through the junction. The RuvB hcxamers are shown in cross-sections, so that the DNA threading through these complexes can be seen. (Source. From Varnada IC et at. Crystal structure of the RuvA RuvB complex. Mol Cell 10 677, fig. 4.)

DNA junctions made by RecA in vitro. Genetic evidence indicates that it functions in concert with RuvA and RuvB.

Resolution by RuvC occurs when RuvC recognizes the Holliday junction (likely in a complex with RuvA and RuvB) and specifically nicks two of the homologous DNA strands that have the same polarity. This cleavage results in DNA ends that terminate with 5' phosphates and 3'OH groups that can be directly joined by DNA ligase. Depending on which pair of strands is cleaved by RuvC, the resulting ligated recombination products will be of either the "splice" (crossover) or "patch" (non-crossover) type. The structure of RuvC and a model schematic proposing how it may interact with junction DNA are shown in Figure 10-13.

Despite recognizing a structure rather than a specific sequence, RuvC cleaves DNA with modest sequence specificity. Cleavage takes place only at sites conforming to the consensus 5'A/T-T-T-G/C. Cleavage occurs after the second T in this sequence. Sequences with this consensus are found frequently in DNA, averaging once every 64 nucleotides. This modest sequence selectivity ensures that at least some branch migration occurs before resolution. Without this sequence selectivity, RuvC might simply cleave Holliday junctions as soon as they are formed, thereby restricting the region of DNA that participates in strand exchange.

FIGURE 10-13 High resolution structure of the RuvC resolvase and schematic model of the RuvC dimer bound to Holliday junction ON A. (a) The crystal structure of the RuvC protein (Ariyoshi M.. vassyfyev D.C, iwasaki H, Nakamura H, Shinagawa K, and Monkawa K, 1994 Cdl 78:1065- 1072.) Image prepared with BobScnpt, MolScrtpt, and Raster 3D. (b) Model for binding oi a RuvC dimer to a Holliday |unction. Notice how, in this model, a dimer of RuvC can bind the Holliday ¡unction and introduce symmetrical cleavages into the two identical DNAstrands. (Source: Rafferty J.B. et al 1996. Crystal structure of DMA recombination protein RuvA. 5acnce 2/4: fig. lb, p. 416, fig. 3e. p. 418. Copyright © 1996 American Association for the Advancement of Science. Reprinted with permission.)

FIGURE 10-13 High resolution structure of the RuvC resolvase and schematic model of the RuvC dimer bound to Holliday junction ON A. (a) The crystal structure of the RuvC protein (Ariyoshi M.. vassyfyev D.C, iwasaki H, Nakamura H, Shinagawa K, and Monkawa K, 1994 Cdl 78:1065- 1072.) Image prepared with BobScnpt, MolScrtpt, and Raster 3D. (b) Model for binding oi a RuvC dimer to a Holliday |unction. Notice how, in this model, a dimer of RuvC can bind the Holliday ¡unction and introduce symmetrical cleavages into the two identical DNAstrands. (Source: Rafferty J.B. et al 1996. Crystal structure of DMA recombination protein RuvA. 5acnce 2/4: fig. lb, p. 416, fig. 3e. p. 418. Copyright © 1996 American Association for the Advancement of Science. Reprinted with permission.)

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