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Dec 26, 2013

Flip On, Flip Off: Tet/DNA Epigenetic Mechanism Uncovered

Flip On, Flip Off: Tet/DNA Epigenetic Mechanism Uncovered

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  • Certain enzymes wrangle with DNA, imposing structural changes that change how DNA is expressed, while preserving the underlying genetic code. A family of such enzymes, called Tet, has aroused interest in recent years. Tet enzymes, researchers have found, help stem cells maintain their multipotent state. Tet enzymes are also involved in early embryonic and brain development and in cancer.

    Now, thanks to scrutiny of a Tet enzyme by means of X-ray crystallography, some of Tet’s structural details have been revealed. These details show how the enzyme interacts with its target DNA, bending the double helix and flipping out the base that is to be modified.

    In a study led by scientists at Emory University School of Medicine, a particular kind of Tet from a single-celled organism was selected to balance ease of crystallization with resemblance to mammalian forms of the enzyme. The organism, known as Naegleria gruberi, is found in soil or fresh water, and it can take the form of an amoeba or a flagellate. Most relevant to the Emory study, it produces a Tet enzyme that is smaller and simpler than mammalian forms, yet still, in the scientist’s words, “shares significant sequence conservation with mammalian Tet1.”

    The Tet protein from N. gruberi (NgTet1) also acts very much like mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC).

    5-mC is generally found on genes that are turned off, or occur on repetitive regions of the genome. 5-mC helps shut off genes that aren’t supposed to be turned on (depending on the cell type) and changes in 5-mC’s distribution underpin a healthy cell’s transformation into a cancer cell.

    In contrast to 5-mC, 5-hmC appears to be enriched on active genes, especially in brain cells. Having a Tet enzyme form 5-hmC seems to be a way for cells to erase or at least modify the “off” signal provided by 5-mC, although the functions of 5-hmC are an active topic of investigation.

    The Emory-based scientists published the results of the study December 25 in Nature, in an article entitled “Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.” The article described the examination of NgTet1 in complex with DNA containing a 5mCpG site, as well as the determination of a crystal structure. This structure, asserted the authors, reveals that NgTet1 uses a base-flipping mechanism to access 5mC: “The DNA is contacted from the minor groove and bent toward the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson–Crick polar hydrogen bonds, and van der Waals' interactions specific for 5mC.”

    The researchers were led by Xiaodong Cheng, Ph.D., a professor of biochemistry at Emory and a Georgia Resarch Alliance Eminent Scholar. “This base flipping mechanism is also used by other enzymes that modify and repair DNA, but we can see from the structure that the Tet family enzymes interact with the DNA in a distinct way," said Dr. Cheng.

    This point was developed in the Nature article by comparing NgTet1 with AlkB, a DNA/RNA repair enzyme found in bacteria, and ABH2, an AlkB homologue found in humans. Like DNA methyltransferases and DNA base excision repair enzymes, NgTet1 and AlkB (and ABH2) use a base-flipping mechanism to access the DNA bases where modification or repair occurs. Despite this similarity, the enzymes do not act alike when fastening themselves to DNA: “The most striking difference between NgTet1 and AlkB is that the bound DNA molecules lie nearly perpendicular to each other relative to the proteins.” To explain this difference, the authors noted that whereas AlkB recognizes a damaged base pair, NgTet1 recognizes a normal Watson–Crick base pair during the initial protein–DNA encounter.

    These findings may help scientists understand how Tet enzymes are regulated and look for drugs that manipulate them. For example, alterations of the Tet enzymes have been found in forms of leukemia, so having information on the enzymes’ molecular structure could help scientists design drugs that interfere with the aberrant forms.



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