Exon shuffling

Molecular mechanism for the formation of new genes

Another mechanism through which exon shuffling occurs is by the usage of helitrons. Helitron transposons were first discovered during studies of repetitive DNA segments of rice, worm and the thale crest genomes. Helitrons have been identified in all eukaryotic kingdoms, but the number of copies varies from species to species. Helitron encoded proteins are composed of a rolling circle (RC) replication initiator (Rep) and a DNA helicase (Hel) domain. The Rep domain is involved in the catalytic reactions for endonucleolytic cleavage, DNA transfer and ligation. In addition this domain contains three motifs. The first motif is necessary for DNA binding. The second motif has two histidines and is involved in metal ion binding. Lastly the third motif has two tyrosines and catalyzes DNA cleavage and ligation. There are three models of gene capture by helitrons: the 'read through" model 1 (RTM1), the 'read through" model 2 (RTM2) and a filler DNA model (FDNA). According to the RTM1 model an accidental "malfunction" of the replication terminator at the 3' end of the Helitron leads to transposition of genomic DNA. It is composed of the read through Helitron element and its downstream genomic regions, flanked by a random DNA site, serving as a "de novo" RC terminator. According to the RTM2 model the 3' terminus of another Helitron serves as an RC terminator of transposition. This occurs after a malfunction of the RC terminator. Lastly in the FDNA model portions of genes or non coding regions can accidentally serve as templates during repair of ds DNA breaks occurring in helitrons. Even though helitrons have been proven to be a very important evolutionary tool, the specific details for their mechanisms of transposition are yet to be defined. An example of evolution by using helitrons is the diversity commonly found in maize. Helitrons in maize cause a constant change of genic and nongenic regions by using transposable elements, leading to diversity among different maize lines.

Long terminal repeat (LTR) retrotransposons are part of another mechanism through which exon shuffling takes place. They usually encode two open reading frames (ORF). The first ORF named gag is related to viral structural proteins. The second ORF named pol is a polyprotein composed of an aspartic protease (AP)which cleaves the polyprotein, an Rnase H (RH) which splits the DNR RNA hybrid, a reverse transcriptase (RT) which produces a cDNA copy of the transposons RNA and a DDE integrase which inserts cDNA into the host's genome. Additionally LTR retrotransponsons are classified into five subfamilies: Ty1/copia, Ty3/gypsy, Bel/Pao, retroviruses and endogenous retroviruses. The LTR retrotransponsons require an RNA intermediate in their transposition cycle mechanism. Retrotransponsons synthesize a cDNA copy based on the RNA strand using a reverse transcriptase related to retroviral RT. The cDNA copy is then inserted into new genomic positions to form a retrogene. This mechanism has been proven to be important in gene evolution of rice and other grass species through exon shuffling.

DNA transposon with Terminal inverted repeats (TIRs) can also contribute to gene shuffling. In plants, some non autonomous elements called Pack TYPE can capture gene fragments during their mobilization. This process appears to be mediated by acquisition of genic DNA residing between neighbouring Pack TYPE transposons and its subsequent mobilization.

Lastly, illegitimate recombination (IR) is another of the mechanisms through which exon shuffling occurs. IR is the recombination between short homologous sequences or nonhomologous sequences. There are two classes of IR: The first corresponds to errors of enzymes which cut and join DNA (i. e., DNases.) This process is initiated by a replication protein which helps generate a primer for DNA synthesis. While one DNA strand is being synthesized the other is being displaced. This process ends when the displaced strand is joined by its ends by the same replication protein. The second class of IR corresponds to the recombination of short homologous sequences which are not recognized by the previously mentioned enzymes. However, they can be recognized by non specific enzymes which introduce cuts between the repeats. The ends are then removed by exonuclease to expose the repeats. Then the repeats anneal and the resulting molecule is repaired using polymerase and ligase.