The authors note that PT modifications, which replace a non-bridging phosphate oxygen with sulphur, were only recently discovered to occur naturally in bacteria. (PT modifications are used by scientists to stabilize synthetic DNA molecules against nuclease degradation.) Today, these modifications have been seen in more than 200 bacteria and archaea, but the detailed genome-wide distribution and biological functions have not been clear.
To look at these events across whole genomes, the scientists used SMRT Sequencing, which can distinguish PT modifications as the polymerase is sequencing DNA. They studied Escherichia coli B7A, which uses the DndF-H proteins known to be associated with PT modifications, as well as Vibrio cyclitrophicus FF75, which lacks those proteins. The PacBio® RS II was used to fully sequence each genome and to assess PT modifications across the genomes.
The scientists found that in E. coli, PT modifications occur on both strands of a particular motif, but only 12 percent of possible motif sites were modified. In V. cyclitrophicus, the modifications are seen only on one DNA strand at CpsCA sequence contexts, but still in just 14 percent of possible sites. The authors also described an iodine-cleavage method in conjunction with Illumina® sequencing which was used to cross-validate the findings; however, that method requires both DNA strands to be modified so was only applied to the E. coli case. “The results raise questions about how Dnd modification proteins (DndA-E) select their DNA targets,” the authors write. “Emerging evidence suggests that DndD is a DNA nicking enzyme and that DndE binds selectively to nicked DNA, with both activities critical to incorporation of PT into the DNA backbone.”
The partial modification seen in both bacteria suggests that overexpression of DndA-E proteins could increase the levels of PT modifications, according to the paper. “These results point to a novel [restriction-modification] system involving site-specific PT modifications without a predictable consensus beyond four nucleotides and with partial modification of sites in the presence of a restriction activity,” the scientists report.
“Such consistency for two bacteria in which PT has very different functions points to a conserved mechanism of DNA target selection by the DNA-modifying DndA-E proteins, a mechanism that we have shown likely involves direct interaction of the modifying proteins with the consensus sequence,” the authors conclude.