R-057. Contribution of Transposase to RecA-Dependent Gene Duplication

A. Reams, J. Roth;
Univ. of California, Davis, CA.

Tandem gene duplications are probably the most common of all genetic changes. Duplications of particular regions are carried by up to 3% of cells in an unselected bacterial population and are common polymorphisms in the human genome. Since duplications are known to arise by exchanges between separated large direct repeats (>1 kb), they are expected to form by homologous recombination. It was surprising that a recA mutant showed only two fold lower initial duplication formation rate for many chromosomal regions of Salmonella enterica. Thus duplications appear to form by both RecA-dependent and -independent routes. A higher RecA dependence was seen on plasmid F’128 where the formation rate of lac duplications was 15-fold lower in the absence of RecA. Our goal was to determine the factors involved in these RecA-dependent lac duplications. All of the RecA-dependent and most of the RecA-independent lac duplications arose by homologous exchanges between two copies of IS3 (1258 bp), which are separated by 131 kb and flank the plasmid lac region. Surprisingly these exchanges were dependent on the IS3 transposase, but did not involve transposition. Instead the IS3 transposase stimulated formation of standard tandem duplications of the entire region between the IS3 elements leaving a copy of IS3 at the duplication join point. The formation rate of RecA-dependent duplications increased with expression of the plasmid transfer (tra) operon, which encodes ability of the plasmid to produce single strand copies of itself and transfer them to a recipient cell. It is proposed that RecA and Tra functions simulate formation of plasmid dimers. When F’128 forms a plasmid dimer, with two IS3 elements in each copy, some resolution events are expected to form a duplication of the region between IS elements. We propose IS3 transposase acts to form and/or resolve these dimers, catalyzing exchanges between copies of IS3. This activity may normally act on cointegrate intermediates made during IS3 transposition. Thus the duplication is generated through the resolution of a plasmid dimer whose formation and/or resolution are stimulated by RecA, Tra, and IS3 transposase.