Extensive, Nonrandom Diversity of Excision Footprints Generated by Ds-Like Transposon Ascot-1Suggests New Parallels with V(D)J Recombination

Abstract

Upon insertion, transposable elements can disrupt or alter gene function in various ways. Transposons moving through a cut-and-paste mechanism are in addition often mutagenic when excising because repair of the empty site seldom restores the original sequence. The characterization of numerous excision events in many eukaryotes indicates that transposon excision from a given site can generate a high degree of DNA sequence and phenotypic variation. Whether such variation is generated randomly remains largely to be determined. To this end, we have exploited a well-characterized system of genetic instability in the fungus Ascobolus immersus to perform an extensive study of excision events. We show that this system, which produces many phenotypically and genetically distinct derivatives, results from the excision of a novel Ds-like transposon, Ascot-1, from the spore color gene b2. A unique set of 48 molecularly distinct excision products were readily identified from a representative sample of excision derivatives. Products varied in their frequency of occurrence over 4 orders of magnitude, yet most showed small palindromic nucleotide additions. Based on these and other observations, compelling evidence was obtained for intermediate hairpin formation during the excision reaction and for strong biases in the subsequent processing steps at the empty site. Factors likely to be involved in these biases suggest new parallels between the excision reaction performed by transposons of the hAT family and V(D)J recombination. An evaluation of the contribution of small palindromic nucleotide additions produced by transposon excision to the spectrum of spontaneous mutations is also presented.Transposons are ubiquitous components of both prokaryotic and eukaryotic genomes, contributing to structural organization, gene activity, and evolution. Two classes of transposons have been distinguished according to their modes of transposition (12). Class I transposons, or retroelements, move by reverse transcription of their RNA, whereas class II transposons are mobilized by either excision/reinsertion or cointegration. Although class I and class II transposons generate variability primarily by their insertion into host DNA, class II elements that move by a cut-and-paste mechanism often produce additional variability at a given site as a result of the excision footprint left at that site. Understanding how these footprints are formed is therefore essential for the proper evaluation of the full mutational impact of the class II transposons.The excision reaction can be separated into two steps, double-strand break formation at both ends of the transposon and repair of the gapped molecule. In vivo and in vitro studies performed with bacterial and animal DNA transposons have shown that all transposases examined cleave precisely at the 3ЈOH ends of the element, which serve for the subsequent strand transfer reaction. On the other hand, the position of the 5Ј cleavage required for the excision of the element need not occur precisely at the tran...

Microhomology-Dependent End Joining and Repair of Transposon-Induced DNA Hairpins by Host Factors in Saccharomyces cerevisiae

“…Similar predominant products have been reported for Ascot excision in the fungus Ascobolus immersus (10). A predominant Ac/Ds footprint also arises in yeast, apparently through a combination of the length and the distance from the broken DNA ends of the microhomology (see Discussion).…”

Hairpin Formation in Tn5 Transposition

“…5Ј nicks are made at the phosphodiester bond one base outside the transposon. Interestingly, the released 3ЈOH does not attack the phosphodiester bond directly oppo-site, but rather attacks the phosphodiester bond immediately 5Ј to it (31). This is essentially the same as the imprecise hairpin formation that we found in Tn5.…”

Perspective: Transposable Elements, Parasitic Dna, and Genome Evolution

“…The excision process may result in either the addition of new sequences or deletion of host sequences flanking the insertion site (Schiefelbein et al 1988). A particularly interesting recent example of sequence addition with potentially important evolutionary implications is provided by the Ds-like transposon Ascot-1 in the fungus Ascobolus immersus (Colot et al 1998). A study of excision products of this element in a spore-color gene revealed small palindromic nucleotide additions in many of them.…”