DNA Methylation and Hydroxymethylation

5-methylcytosine and 5-hydroxymethylcytosine Exert Opposite Forces on Base Pairing of DNA Double Helix

DNA methylation     DNA base pairing governs the fundamental function of DNA in life. Importantly, annealing and unwinding of base-paired double helical DNA strands are essential for DNA replication and transcription processes. Moreover, epigenetic DNA base modifications have become recognized to be involved in regulation of DNA at all levels in higher organisms. Our recent research into DNA base modifications has shown that 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) modifications dramatically change the properties of C:G base pairing. In contrast to the 5-mC:G pairing, which increases the base pairing stability relative to normal C:G pairing, we find that 5-hmC:G base pairing greatly decreases stability relative to both C:G and 5-mC:G base pairing. It is evident that cytosine epigenetic modifications provide another layer of hidden codes, which serve as a “lock”, neutral and “unlock” mechanism on DNA beyond the canonical genetic codes. We call this the Double Helix Epigenetic Switch.

DNA is the blueprint for life, coding all of the genes needed in each cell within each tissue in all organisms on Earth. It has been over half a century since the discovery of the DNA double helix and uncovering of genetic codes. In the last decade, the development of epigenetic understanding has further elucidated some fundamental mechanisms of how genes are organized, regulated and inherited through elaborated epigenetic regulation mechanisms. (In addition, the century old debate on nature versus nurture has finally begun to converge into a more complete picture of biology, where genetics and epigenetics are both considered. It is now clear that both nature and nurture are important). Cytosine modifications in both 5-mC and 5-hmC are two important epigenetic markers and their involvement in gene regulation has been intensively studied in the last decade. Although fundamental A:T and C:G base pairings are well known for the DNA double helix structure, the direct biochemical effects of epigenetically modified bases of 5-mC and 5-hmC on DNA has not been thoroughly investigated. Here we report the 5-mC and 5-hmC base modification effects on C:G base pairing and the overall effects on dsDNA stability.

High resolution melting (HRM) analysis was used to measure the dsDNA stability. This analysis directly measures DNA as either dsDNA (base-paired) or single stranded (denatured) status. This was used as a measurement of DNA stability for different cytosine modifications in a 897bp DNA fragment (5-methylcytosine & 5-hydroxymethylcytosine DNA Standard Set, D5405, Zymo Research Corp.) with relative evenly distributed G, A, T and C. The C was either 100% native C, or 100% 5-mC or 5-hmC.

The 5-mC containing DNA showed a dramatic increase in DNA melting temperature, on the other hand, the 5-hmC showed a dramatic decrease in DNA melting temperature (Fig 1A). When the 50% DNA melting point was used for measurement, 5-mC could increase the effective DNA denaturation temperature by 6°C while 5-hmC decreased the effective DNA denaturation temperature by over 2°C in relation to native C. When measuring 5-hmC vs 5-mC, the melting temperature difference was shown to be over 8°C for the same DNA (Fig 1B). The above observed results were demonstrated using a relatively large DNA fragment (897bp) and represented the collective effect of the whole fragment.

DNA melting curve




Figure 1. 5-Hydroxymethylcytosine decreases thermodynamic stability of DNA. Procedure: (A) Melting curves of DNA standards containing 100% of their cytosine as either unmodified cytosine (C), 5-methylcytosine (5-mC), or 5- hydroxymethylcytosine (5-hmC) were analyzed by high resolution melting (HRM). Samples were done in triplicate and averages were plotted. (B) Tm’s were calculated by finding the 50% relative fluorescence levels.






Next, we measured the single cytosine base modification effect on dsDNA stability. To do this, a synthetic 52bp template was designed with a modified C in the middle (Fig 2A). In this set up, the DNA melting temperature changes will result from the effect of the single modified base. As shown in Fig 2B, the effect of the DNA melting temperature could be observed reproducibly, even on a single base modification. This demonstrates that the modifications are affecting the strength of the C:G base pairing. Clearly the 5-hmC:G bond is noticeably weaker than the 5-mC:G bond and the normal C:G bond strength is somewhere in between. This and several other experiments (data not shown here) showed similar results, all of which concluded that the 5-mC increases the dsDNA stability.

5hmc decreases thermostability





Figure 2. 5-Hydroxymethylcytosine decreases thermodynamic stability of DNA Procedure: Template was created by primer extension with a dNTP mix containing either cytosine, 5-methylcytosine, or 5-hydroxymethylcytosine. (A) emplates were designed to incorporate either cytosine on the extended strand. Template strand (bottom strand 52mer) and elongation primer (italicized bold 24mer). (B) Melting curves were analyzed by high resolution melting (HRM). Tm’s were calculated by finding the 50% relative fluorescence levels.



Taken together, these results present a unique view of the dynamics of epigenetic modifications. The cytosine modifications not only cause structural changes on the DNA backbone, which may affect the protein binding directly due to the changed chemical structure, but these modifications can also affect the stability of the double helix directly. It is well known that DNA unwinding is an essential step in transcription initiation and DNA replication. It is conceivable that the cytosine mC and hmC modifications also serve as a DNA intrinsic ”molecular switch.” We call this the Double Helix Epigenetic Switch for its potential to be in a locked, neutral and unlocked status. Thus, cytosine epigenetic modifications give dsDNA another coding dimension beyond the primary code. Together, genetic and epigenetic information render dsDNA into life’s blueprint.


Original Article:

R. Leavitt. 5-methylcytosine and 5-hydroxymethylcytosine Exert Opposite Forces on Base Pairing of DNA Double Helix. Peanuts Biotechnical Newsletter. Zymo Research. pg. 6-7; (http://www.zymoresearch.com/pdf/publications/2015peanuts2.pdf)

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Larry Jia

Larry Jia