With a finding published online in the July 21, 2011 issue of the journal Science, researchers from the University of North Carolina at Chapel Hill School of Medicine and Gillings School of Global Public Health have discovered the seventh and eighth bases of DNA. These last two bases – called 5-formylcytosine and 5-carboxylcytosine – are actually versions of cytosine that have been modified by Tet proteins, molecular entities thought to play a role in DNA demethylation and stem cell reprogramming. The discovery could advance stem cell research by giving a glimpse into the DNA changes (such as the removal of chemical groups through demethylation) that could reprogram adult cells to make them act like stem cells.
"Before we can grasp the magnitude of this discovery, we have to figure out the function of these new bases," said senior study author Dr. Yi Zhang, Kenan Distinguished Professor of Biochemistry and Biophysics at UNC and an investigator of the Howard Hughes Medical Institute at the Lineberger Comprehensive Cancer Center. "Because these bases represent an intermediate state in the demethylation process, they could be important for cell reprogramming and cancer, both of which involve DNA demethylation." Much is known about the "fifth base," 5-methylcytosine, which arises when a chemical tag or methyl group is tacked onto a cytosine. This methylation is associated with gene silencing, as it causes the DNA's double helix to fold even tighter upon itself. Last year, Dr. Zhang's group reported that Tet proteins can convert 5 methylC (the fifth base) to 5 hydroxymethylC (the sixth base) in the first of a four-step reaction leading back to bare-boned cytosine. But try as they might, the researchers could not continue the reaction on to the seventh and eighth bases, called 5-formylC and 5-carboxyC. The problem, they eventually found, was not that Tet wasn't taking that second and third step, it was that their experimental assay was not sensitive enough to detect it. Once they realized the limitations of the assay, they redesigned it and were able to detect the two newest bases of DNA with electrophoresis, but reviewers wanted more detailed chemical analyses. The Zang laboratory contacted Dr. James Swenberg, Kenan Professor of Environmental Sciences and Engineering, who introduced them to Leonard Collins from the Biomarker Mass Spectrometry Facility Core. Together, they quickly developed advanced mass spectrometry methods to fully characterize the new bases and demonstrated the presence of as little as 5 fmol of the minor bases in stem cells and mouse tissue DNA. Other study co-authors include UNC Lineberger Comprehensive Cancer Center postdoctoral research associates Dr. Shinsuke Ito, Dr. Li Shen and Dr. Susan C. Wu.