Multiple Pathways of Duplication Formation with and Without Recombination (RecA) in Salmonella enterica

Abstract

Duplications are often attributed to "unequal recombination" between separated, directly repeated sequence elements (.100 bp), events that leave a recombinant element at the duplication junction. However, in the bacterial chromosome, duplications form at high rates (10 23 -10 25 /cell/division) even without recombination (RecA). Here we describe 1800 spontaneous lac duplications trapped nonselectively on the low-copy F9 128 plasmid, where lac is flanked by direct repeats of the transposable element IS3 (1258 bp) and by numerous quasipalindromic REP elements (30 bp). Duplications form at a high rate (10 24 /cell/division) that is reduced only about 11-fold in the absence of RecA. With and without RecA, most duplications arise by recombination between IS3 elements (97%). Formation of these duplications is stimulated by IS3 transposase (Tnp) and plasmid transfer functions (TraI). Three duplication pathways are proposed. First, plasmid dimers form at a high rate stimulated by RecA and are then modified by deletions between IS3 elements (resolution) that leave a monomeric plasmid with an IS3-flanked lac duplication. Second, without RecA, duplications occur by singlestrand annealing of DNA ends generated in different sister chromosomes after transposase nicks DNA near participating IS3 elements. The absence of RecA may stimulate annealing by allowing chromosome breaks to persist. Third, a minority of lac duplications (3%) have short (0-36 bp) junction sequences (SJ), some of which are located within REP elements. These duplication types form without RecA, Tnp, or Tra by a pathway in which the palindromic junctions of a tandem inversion duplication (TID) may stimulate deletions that leave the final duplication. G ENE duplications are among the first mutations for which a physical basis was known. The Bar-Eye mutation of Drosophila was discovered in 1914 (Tice 1914) and shown genetically and cytologically in 1936 to be a duplication (Bridges 1936;Muller 1936)-well before the Watson-Crick model for DNA. The attached-X mutation was discovered even earlier and is essentially an inversion duplication (Morgan 1925). Recently, duplications have been shown to be exceedingly common polymorphisms in human populations (Conrad et al. 2010) and somatic amplifications play an important role in origins of malignancy and resistance to cancer therapies (Lu et al. 2008;Zhang et al. 2009;Conrad et al. 2010). Gene duplications also play a major role in the evolution of new genes (Ohno 1970;Bergthorsson et al. 2007). Despite the high frequency and biological importance of duplications, the process by which they form remains uncertain.Distinct models have been suggested for duplication formation in various genetic systems. In Drosophila, duplications often have transposable elements between copies (Green 1985;Tsubota et al. 1989). In humans, duplications most commonly arise between extensive sequence repeats (Redon et al. 2006) and are sometimes promoted by palindromic sequence elements (Conrad et al. 2010). A major question is how...

Bacterial Genome Instability

“…They are hot spots for specific transpositions (72)(73)(74)(75), and they have been found at the junctions of RecA-dependent and RecA-independent duplications (76,77).…”

“…6 and 7) (for more details, see references 325, 333, 335, 362, 366, and 368). Importantly, illegitimate events create novel junction sequences, whereas the misaligned homologous recombination events do not (77).…”

“…7C) (77). Models for how these structures can be generated have been proposed and involve various strand slippage reactions during DNA replication and DNA repair (77,385,386).…”

“…7C) (77). Models for how these structures can be generated have been proposed and involve various strand slippage reactions during DNA replication and DNA repair (77,385,386). The structure formed consists of two overlapping DNA palindromes and is itself likely to be prone to hairpin structure-stimulated strand slippage that reduces the symmetry of the palindrome centers.…”

Rapid Evolution of Citrate Utilization by Escherichia coli by Direct Selection Requires citT and dctA

“…Increased expression of the CitT transporter allowed minimal but sufficient access to citrate and a low level of cell division. Gene amplifications are the most common mutations identified (28) and set the stage for subsequent promoter capture recombinatorial events. Thus, Cit ϩ cells amplified for citT, such as the weakly Cit ϩ DV130 cells, transitioned by citT promoter capture and dctA amplification to DV133, the strongly Cit ϩ derivative.…”