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Figure 3 Double-strand break repair by recombination-directed replication. A. D loops formed by the combination of RecA, RecBCD and SSB are the substrate for both a nonspecific strand-displacement DNA synthesis reaction (no PriA) and for assembly of a bona fide replication fork (with PriA).
B and C. Omission experiments showing the requirements for the various components of the reaction. |
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To model the intersection between recombination and replication predicted by the phenotypes of the priA null mutant, we reconstituted the initial steps of double-strand break repair (DSBR) mediated by recombination-dependent replication (RDR). This system used an 800mer linear DNA carrying a recombination-enhancing 
site as the broken sister arm of the chromosome and a supercoiled plasmid as the intact sister arm of the chromosome (Figure 3). Recombinant joint molecules are formed by the action of RecA, RecBCD, and SSB and were shown to be, in concerted reactions, the substrate for 2 different replication reactions. The combination of DNA gyrase-mediated maintenance of net negative supercoiling of the plasmid template and the cooperative binding of the single-stranded DNA-binding protein (SSB) supported a generalized strand-displacement DNA synthesis reaction from the recombinant joint.
Here the 3
-OH of the invading strand could be used as a primer by any polymerase to form full length single-stranded product (Figure 3). This observation implied that despite the fact that the heteroduplex formed by the invading strand and the plasmid template strand in the D-loop was nominally engaged within a RecA filament, the polymerase had access to the 3-OH of the invading strand. We therefore investigated this aspect of the reaction and found that DNA replication was inhibited if disassembly of the RecA filament was prevented. Thus, the dynamic nature of the RecA filament allows it to clear from the region of heteroduplex in a joint molecule. This clearance, in turn, allows subsequent reactions to occur on the joint molecules, permitting a choice of enzymatic pathway for resolution.
It is particularly interesting that because RecA filaments assemble and disassemble in the same direction, 5
3, clearance occurs in a manner that specifically encourages subsequent DNA replication that uses the 3-end of the invading strand as a primer for leading-strand synthesis. Such a generalized strand-displacement reaction provides a plausible mechanism for the DSB-induced replication that has been observed in yeast and human cells. On the other hand, the relatively promiscuous nature of this reaction suggests that its limitation would be advantageous to the cell. In E. coli, we found that this limiting function is supplied by PriA.
In the presence of the restart primosomal proteins (PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG), RecA, RecBCD, SSB, the DNA polymerase III holoenzyme (Pol III HE, the replicative polymerase), and DNA gyrase, bona fide replication forks were formed on the joint molecules, resulting in the production of a late replicative intermediate (LRI) with a continuous leading strand and discontinuous lagging strand (Figure 3). Completed molecules could be formed if topoisomerase IV was added to the reaction mixture. RDR was dependent on all of the restart primosomal proteins except PriC. We now understand the reason for this lack of correspondence with the original 
X system, in that the restart primosomal proteins actually encompass 2 distinct replisome-loading systems (see Multiple Pathways of Replication Restart).
