Genes VII
14.16 Retrieval systems in E. coli |
| Key terms defined in this section |
| Single-strand exchange is a reaction in which one of the strands of a duplex of DNA leaves its former partner and instead pairs with the complementary strand in another molecule, displacing its homologue in the second duplex. |
| Figure 14.35 An E. coli retrieval system uses a normal strand of DNA to replace the gap left in a newly synthesized strand opposite a site of unrepaired damage. |
Retrieval systems have variously been termed "post Vreplication repair," because they function after replication, or "recombination-repair," because the activities overlap with those involved in genetic recombination. Such systems are effective in dealing with the defects produced in daughter duplexes by replication of a template that contains damaged bases. An example is illustrated in Figure 14.35.
Consider a structural distortion, such as a pyrimidine dimer, on one strand of a double helix. When the DNA is replicated, the dimer prevents the damaged site from acting as a template. Replication is forced to skip past it.
DNA polymerase probably proceeds up to or close to the pyrimidine dimer. Then the polymerase ceases synthesis of the corresponding daughter strand. Replication restarts some distance farther along. A substantial gap is left in the newly synthesized strand.
The resulting daughter duplexes are different in nature. One has the parental strand containing the damaged adduct, facing a newly synthesized strand with a lengthy gap. The other duplicate has the undamaged parental strand, which has been copied into a normal complementary strand. The retrieval system takes advantage of the normal daughter.
The gap opposite the damaged site in the first duplex is filled by stealing the homologous single strand of DNA from the normal duplex. Following this single-strand exchange, the recipient duplex has a parental (damaged) strand facing a wild Vtype strand. The donor duplex has a normal parental strand facing a gap; the gap can be filled by repair synthesis in the usual way, generating a normal duplex. So the damage is confined to the original distortion (although the same recombination-repair events must be repeated after every replication cycle unless and until the damage is removed by an excision repair system).
| Figure 14.9 RecBCD nuclease approaches a chi sequence from one side, degrading DNA as it proceeds; at the chi site, it makes an endonucleolytic cut, loses RecD, and retains only the helicase activity. |
| 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. |
| 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 principal pathway for recombination-repair in E. coli is identified by the rec genes, whose activities in recombination per se we discussed earlier (see Figure 14.9, Figure 14.10, Figure 14.11). In E. coli deficient in excision repair, mutation in the recA gene essentially abolishes all the remaining repair and recovery facilities. Attempts to replicate DNA in uvr VrecA V cells produce fragments of DNA whose size corresponds with the expected distance between thymine dimers. This result implies that the dimers provide a lethal obstacle to replication in the absence of RecA function. It explains why the double mutant cannot tolerate >1 V2 dimers in its genome (compared with the ability of a wild-type bacterium to handle as many as 50).
One rec pathway involves the recBC genes, and is well characterized; the other involves recF, and is not well defined. The ability of RecA to exchange single strands allows it to perform the retrieval step in Figure 14.35. Nuclease and polymerase activities then complete the repair action.
The designations of repair and recombination genes are based on the phenotypes of the mutants; but sometimes a mutation isolated in one set of conditions and named as a uvr locus turns out to have been isolated in another set of conditions as a rec locus. This uncertainty makes an important point. We cannot yet define how many functions belong to each pathway or how the pathways interact. The uvr and rec pathways are not entirely independent, because uvr mutants show reduced efficiency in recombination Vrepair.
We must expect to find a network of nuclease, polymerase, and other activities, constituting repair systems that are partially overlapping (or in which an enzyme usually used to provide some function can be substituted by another from a different pathway).