Multicopy single-stranded DNA

[[File:Myxobacterial msDNA. svg|thumb|245px|msDNA from Stigmatella aurantiaca compared with msDNA from the closely related Myxococcus xanthus. The hypervariable domain in the DNA sequence is shaded gray. The highly conserved AGC RNA sequence including the branch G is shaded pink. An RNA cleavage site between precursor and product forms of msDNA is indicated by a red triangle. Redrawn from Dhundale et al.

Before the discovery of msDNA in myxobacteria, a group of swarming, soil dwelling bacteria, it was thought that the enzymes known as reverse transcriptases (RT) existed only in eukaryotes and viruses. The discovery led to an increase in research of the area. As a result, msDNA has been found to be widely distributed among bacteria, including various strains of Escherichia coli and pathogenic bacteria. Further research discovered similarities between HIV encoded reverse transcriptase and an open reading frame (ORF) found in the msDNA coding region. Tests confirmed the presence of reverse transcriptase activity in crude lysates of retron containing strains. Although an RNase H domain was tentatively identified in the retron ORF, it was later found that the RNase H activity required for msDNA synthesis is actually supplied by the host.

The discovery of msDNA has led to broader questions regarding where reverse transcriptase originated, as genes encoding for reverse transcriptase (not necessarily associated with msDNA) have been found in prokaryotes, eukaryotes, viruses and even archaea. After a DNA fragment coding for the production of msDNA in E. coli was discovered, it was conjectured that bacteriophages might have been responsible for the introduction of the RT gene into E. coli. These discoveries suggest that reverse transcriptase played a role in the evolution of viruses from bacteria, with one hypothesis stating that, with the help of reverse transcriptase, viruses may have arisen as a breakaway msDNA gene that acquired a protein coat. Since nearly all RT genes function in retrovirus replication and/or the movement of transposable elements, it is reasonable to imagine that retrons might be mobile genetic elements, but there has been little supporting evidence for such a hypothesis, save for the observed fact that msDNA is widely yet sporadically dispersed among bacterial species in a manner suggestive of both horizontal and vertical transfer. Since it is not known whether retron sequences per se represent mobile elements, retrons are functionally defined by their ability to produce msDNA while deliberately avoiding speculation about other possible activities.

The function of msDNA remains unknown even though many copies are present within cells. Knockout mutations that do not express msDNA are viable, so the production of msDNA is not essential to life under laboratory conditions. Over expression of msDNA is mutagenic, apparently as a result of titrating out repair proteins by the mismatched base pairs that are typical of their structure. Sequence comparison of msDNAs from Myxococcus xanthus, Stigmatella aurantiaca, and many other bacteria The major msDNAs of M. xanthus and S. aurantiaca, for instance, share 94% sequence homology except within a 19 base pair domain that shares sequence homology of only 42%.

[[File:Msdna synthesis. png|thumb|270px|Proposed mechanism for the synthesis of msDNA. (A) Folding of the primer template RNA into a secondary structure allows the 2' OH group of a specific branching G residue to serve as a primer to initiate cDNA synthesis by the retron reverse transcriptase. (B) Synthesis of cDNA is accompanied by RNase H digestion of the template strand. (C) In the completed msDNA molecule, part of the RNA template remains joined to the 5' end of the cDNA. The RT recognizes specific stem loop structures in the precursor RNA, rendering synthesis of msDNA by the RT highly specific to its own retron. The priming of msDNA synthesis offers a fascinating challenge to our understanding of DNA synthesis. DNA polymerases (which include RT) share highly conserved structural features, which means that their active catalytic sites vary little from species to species, or even between DNA polymerases using DNA as a template, versus DNA polymerases using RNA as a template. The catalytic region of eukaryotic reverse transcriptase comprises three domains termed the "fingers", "palm", and "thumb" which hold the double stranded primer template in a right hand grip with the 3' OH of the primer buried in the active site of the polymerase, a cluster of highly conserved acidic and polar residues situated on the palm between what would be the index and middle fingers. In eukaryotic RTs, the RNase H domain lies on the wrist below the base of the thumb, but retron RTs lack RNase H activity. The nucleic acid binding cleft, extending from the polymerase active site to the RNase H active site, is about 60 Å in length in eukaryotic RTs, corresponding to nearly two helical turns. When eukaryotic RT extends a conventional primer, the growing DNA/RNA double helix spirals along the cleft, and as the double helix passes the RNase H domain, the template RNA is digested to release the nascent strand of cDNA. In the case of msDNA primer extension, however, a long strand of RNA remains attached to the 3' OH of the priming G. Although it is possible to model an RT primer template complex which would make the 2' OH accessible for the priming reaction, further extension of the DNA strand presents a problem: as DNA synthesis progresses, the bulky RNA strand extending from the 3' OH needs somehow to spiral down the binding cleft without being blocked by steric hindrance. To overcome this issue, the msDNA reverse transcriptase clearly would require special features not shared by other RTs.