Expansion of DNA repeats in Escherichia coli: effects of recombination and replication functions.

Duplication or expansion of directly repeated sequence elements is associated with a number of human genetic diseases. To study the mechanisms of repeat expansion, we have developed a plasmid assay in Escherichia coli. Our assay involves two simple repeats of 787 bp in length; expansion to three or more copies of the repeat can be selected by restoration of an intact tetracycline-resistance gene. Expansions occurred at relatively high rates, >10(-5), in the population. Both RecA-dependent recombination and RecA-independent slipped misalignments contributed to the observed expansion events. Mutations that impair DNA polymerase III (DnaE, DnaQ subunits) or the replication fork helicase, DnaB, stimulated both RecA-dependent and RecA-independent expansion events. In these respects, the properties of repeat expansion resemble repeat deletion and suggest that difficulties in DNA replication may trigger both classes of rearrangements. About 20% of the RecA-independent expansion events are accompanied by reciprocal sister-chromosome exchange, producing dimeric plasmids carrying one triplicated and one deleted locus. These products are explained by a model involving misaligned strands across the replication fork. This model predicts that the location of a replication stall site may govern the types of resulting rearrangements. The specific location of such a stall site can also, in theory, account for propensity towards expansion or deletion of repeat arrays. This may have relevance to trinucleotide repeat expansion in human genetic disease.

Tandem repeat recombination induced by replication fork defects in Escherichia coli requires a novel factor, RadC.

DnaB is the helicase associated with the DNA polymerase III replication fork in Escherichia coli. Previously we observed that the dnaB107(ts) mutation, at its permissive temperature, greatly stimulated deletion events at chromosomal tandem repeats. This stimulation required recA, which suggests a recombinational mechanism. In this article we examine the genetic dependence of recombination stimulated by the dnaB107 mutation. Gap repair genes recF, recO, and recR were not required. Mutations in recB, required for double-strand break repair, and in ruvC, the Holliday junction resolvase gene, were synthetically lethal with dnaB107, causing enhanced temperature sensitivity. The hyperdeletion phenotype of dnaB107 was semidominant, and in dnaB107/dnaB+ heterozygotes recB was partially required for enhanced deletion, whereas ruvC was not. We believe that dnaB107 causes the stalling of replication forks, which may become broken and require repair. Misalignment of repeated sequences during RecBCD-mediated repair may account for most, but not all, of deletion stimulated by dnaB107. To our surprise, the radC gene, like recA, was required for virtually all recombination stimulated by dnaB107. The biochemical function of RadC is unknown, but is reported to be required for growth-medium-dependent repair of DNA strand breaks. Our results suggest that RadC functions specifically in recombinational repair that is associated with the replication fork.

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

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.

Enhanced deletion formation by aberrant DNA replication in Escherichia coli.

Repeated genes and sequences are prone to genetic rearrangements including deletions. We have investigated deletion formation in Escherichia coli strains mutant for various replication functions. Deletion was selected between 787 base pair tandem repeats carried either on a ColE1-derived plasmid or on the E. coli chromosome. Only mutations in functions associated with DNA Polymerase III elevated deletion rates in our assays. Especially large increases were observed in strains mutant in dnaQ the epsilon editing subunit of Pol III, and dnaB, the replication fork helicase. Mutations in several other functions also altered deletion formation: the alpha polymerase (dnal;), the gamma clamp loader complex (holC, dnaX), and the beta clamp (dnaN) subunits of Pol III and the primosomal proteins, dnaC and priA. Aberrant replication stimulated deletions through several pathways. Whereas the elevation in dnaB strains was mostly recA- and lexA-dependent, that in dnaQ strains was mostly recA- and lexA-independent. Deletion product analysis suggested that slipped mispairing, producing monomeric replicon products, may be preferentially increased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants. We conclude that aberrant Polymerase III replication can stimulate deletion events through several mechanisms of deletion and via both recA-dependent and independent pathways.