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(Received for publication, September 16, 1994; and in revised form, November 2,
1994) 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.
Volume 270,
Number 2,
Issue of January 13, 1995 pp. 581-588
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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