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FIGURE 10-8 Three views of the RecA filament (a) Electron micrograph of circular DMA molecules that are fully or partially coated with RecA An uncoated DMA molecule is also shown to idustwte how the DNA is elongated upon RecA binding. (Source: Reprinted with permission from Stasiak A and Egelman E.H. 1986. Visualization of Recombination Reactions, p. 265-307, in Genetic Recombination R. Kucherla-paii and G. Smith, eds„ ASM Press. From Figure 3.) (b) A higher resolution view of the filament generated by averaging many EM images, the picture on the left is E coti RecA, whereas the one on the right ts the related strand-exchange protein Rad5l from yeast. (Source: Irrage provided by Edward Egelman, University of vii^inia.) (c) A higher resolution view generated by X-ray civstaSography. Here one turn of the helical filamert is shown from a top dowr, view. Individual subumts are Colored. the red subunit is closest to the viewer (Story RM and Steitz TA 1992. Nature 355 316.) Image prepared with BobScnpt, MoScript, and Raster 3D.

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RecA

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5'l no filament formation active filament formation to coat 3" end of ssDNA

FIGURE 10-9 Polarity of RecA assembly. Note that new subunits of RecA join the filament on the DNA 3' side to an existing subumt much faster than these subunits join on the 5' side. Because of this polarity of assembfy, DMA molecules with 3' ssDNA extensions will be efficiently coated with RecA. In contrast, molecules with 5' ssDNA extensions would not serve as substrates for filament assembfy.

on double-stranded DNA, thus explaining the need for regions of ssDNA iri si rand-exchange substrates. The filament grows by the addition of RecA subunits in the 5' to 3' direction, such that a DNA strand that terminates in 3' ends is most likely to be coated by RecA (Figure 10-9). Note that in the DSB-repair model for recombination, it is DNA molecules with just this structure that participate in strand invasion.

Newly Base-Paired Partners Are Established within the RecA Filament

RecA-catalyzed strand exchange can be divided into distinct reaction stages. First, fhe RecA filament must assemble on one of the participating DNA molecules. Assembly occurs on a molecule containing a region of single-stranded DNA, such as an ssDNA tail. This RecA-ssDNA complex is the active form that participates in the search for a homology. During this search. RecA must "look" for base-pair complementarity between the DNA within the filament and a new DNA molecule.

This homology search is promoted by RecA because the filament structure has two distinct DNA-binding sites; a primary site (bound by the first DNA molecule), and a secondary site (Figure 10-10). This secondary DNA-binding site can be occupied by double-stranded DNA. Binding to this site is rapid, weak, transient and—importantly—independent of DNA sequence. In this way. the RecA filament can bind and rapidly "sample" huge stretches of DNA for sequence homology.

How does the RecA filament sense sequence homology? Details of this mechanism are still not clear. The DNA in the secondary binding site is transiently opened and tested for complementarity with the ssDNA in the primary site. This "testing" is presumably via base-pair-ing interactions, although it occurs initially without disrupting the global base-pairing between the two strands of the DNA in the secondary she, hi support of this idea, experiments suggest that the ini tial alignment may involve base-flipping of some of the bases in the DNA duplex (see Chapter 9 for a discussion of base-flipping during DNA repair). In vitro experiments indicate that a sequence match of just 15 base pairs provides a sufficient signal to the RecA filament that a match has been found, and thereby trigger strand exchange, primary binding site

RecA filament

RecA filament secondary binding site cross-section of single DNA strand bound to RecA protein

FIGURE 10-10 Model of two steps in the search for homology and DNA strand exchange within the RecA filament. Here the RecA filament is represented from a top down view as in Figure 10-6c. the incoming DNA duplex is shown in blue (Source: Adapted from Howard Banders et al 1984. Nature 309: 215- 220. Copyright © 1984 Nature Publishing Group. Used with permission)

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