Slipped misalignment mechanisms of deletion formation: in vivo susceptibility to nucleases

Abstract

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3' exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3' ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.

FIG. 1

Replication misalignment models for deletion formation. Newly replicated DNA is denoted with dashed lines. (A) Simple slipped mispairing involves the dislocation of a nascent strand to mispair with a second copy of a repeated sequence on its template, forming a looped misaligned intermediate. A 3′ end may be transiently unpaired and susceptible to 3′ exonucleases during this process. If replication is completed, a monomeric deletion product will result. (B) Sister chromosome mispairing involves the displacement and mispairing of both nascent strands in a stalled replication fork. This mispairing produces a Holliday junction-like intermediate which may resolve as a crossover between sister chromosomes, producing a dimeric replicon. Alternative resolution may produce monomeric deletion chromosomes.

FIG. 2

Instability of the looped intermediate of simple slipped misalignment. During deletion formation, the mispairing of nascent and template strands produces a loop on the template strand. Migration of the loop within the repeat can destabilize the intermediate by shortening the heteroduplex pairing region adjacent to the nascent 3′ end. Degradation of the 3′ end by DNA polymerase III (DnaQ) can also destabilize the intermediate and abort potential deletion events.