The formation of the 2',5'-phosphodiester linkage in the cDNA priming reaction by bacterial reverse transcriptase in a cell-free system.

Bacterial reverse transcriptase (RT) is responsible for synthesis of multicopy single-stranded DNA (msDNA) consisting of single-stranded DNA linked to an internal guanosine residue of RNA by an unusual 2',5'-phosphodiester linkage. Here we purified a bacterial RT to homogeneity from Escherichia coli harboring the RT gene from retron-Ec73. The purified RT-Ec73 was able to synthesize msDNA in a cell-free system using an RNA template produced in vitro by T7 RNA polymerase. The in vitro synthesized msDNA was released from the template RNA only when treated with yeast debranching enzyme DBR1, a specific nuclease for a 2',5'-phosphodiester linkage. The position of the branching G residue in the template RNA and the DNA sequence of the cell-free product were identical to those of msDNA-Ec73 synthesized in vivo. These results clearly demonstrate that the formation of the 2',5'-phosphodiester linkage in msDNA synthesis is carried out by RT itself.