127 Template-switching during DNA replication

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Short repetitive DNA sequences are frequent sites of genomic rearrangements. Using the bacterium Escherichia coli, we have studied the mechanisms of these frequent events and have proposed that they arise by a template-switch mechanism during DNA replication. We hypothesize that the template-switch mechanism is a replication-gap filling repair mechanism, which, like translesion DNA synthesis, can overcome replication-blocking lesions on the DNA template strand. Both translesion synthesis (TLS) and template-switching are components of what has been termed “post-replication repair.” Tranlesion synthesis has been considered “error-prone” because of its propensity to induce point mutations because of the low fidelity of the TLS polymerases. Template-switching, we propose, comprises what has been considered the “error-free” pathway of post-replication repair. We note that the pathway is not truly error-free, as it is “rearrangement-prone.” Using genetic assays for deletions or expansions at short repeats, we have investigated the properties of the template-switch pathway. Our analysis shows that template-switching frequently occurs, concomitant with sister-chromosome exchange. These crossovers occur independently of the known homologous recombination machinery including RecA. Whereas homologous recombination requires a threshold homology of about 250 bp (below which recombination is inefficient), the template-switching crossovers can occur between microhomologies as low as 25 bp. Blocking replication with the chain-terminator azidothymidine (AZT) stimulates template-switching as do mutations in the DNA polymerase III replisome, consistent with the idea that template-switching occurs as a response to replication gaps. We have identified three proteins whose functions are required for template-switching. Mutants in these functions exhibit enhanced sensitivity to DNA replication inhibitors AZT and hydroxyurea and lower rates of genetic rearrangements. The first template-switch factor is chaperone DnaKJ, identified through a genetic screen. Mutations in dnaK also block the “error-prone” repair pathway, as they exhibit lower rates of DNA polymerase V-dependent mutagenesis induced by UV irradiation. Mutations in the processivity clamp for replication (dnaN) and in one subunit of the clamp loader complex (dnaX) also diminish template-switching. By a programmed translational frameshift, the dnaX gene produces two variants, a larger form required for replication and a shorter form of unknown function. Our recent experiments suggest that the shorter form is required for template-switching. We propose that this short form is used in an alternative clamp loader complex employed during DNA repair.