DNA methylation is the addition of a methyl (-CH<sub>3</sub>) group to the 5 position carbon located at the pyrimidine ring of cytosine(C), and is one of the epigenetic mechanisms that control the function of genes without changing the genetic code. DNA methylation plays an important role in various life phenomena, such as X-chromosome inactivation, gene imprinting, normal ontogeny, and development. In addition, hypermethylation of a promotor in the dense region of cytosine followed by a guanine in the linear sequence of bases is associated with the gene silencing in cancer cells. Therefore, the understanding of DNA methylation and the replication of methylated DNA is mightily important. For this reason, it is essential to understand the binding between DNA polymerases (DNAP) and a methlyated DNA in terms of kinetics and thermodynamics.<br/><br/>Atomic force microscopy (AFM) has the capability of measuring intermolecular forces at the pico-Newton level and scanning surfaces at the nanometer resolution. Single molecule force spectroscopy (SMFS) using AFM has been widely used to observe the biomolecular interactions. In our approach, we employed force-based AFM to study the binding of deoxyguanosine triphosphate (dGTP) immobilized on a glass slide to a DNAP (Human polymerase delta catalytic subunit, POLD1) tethered at an AFM tip. By allowing a template DNA to be captured by the DNAP either C or 5-methylcytosine (5mC) is positioned at the active site. The specific rupture events arising from the binding of complementary bases to the primer/template DNA strand (containing C or 5mC)/DNAP complex were observed. The event frequency within a specific area is dependent upon the concentration of the complex in solution. Also, the frequency is also a function of dGTP in solution. The equilibrium constant could be obtained from such change. It is noteworthy that the thermodynamic information is obtained at the single molecule level. Increasing the contact time between the DNAP and dGTP enhanced the event frequency for the two cases. The relevant binding kinetic rates at the single molecule level are expected to be obtained by plotting the frequency with respect to the contact time.

Recognition of methylated cytosine in a DNA polymerase for dGTP: Retrieving association rate constant at the single molecule level

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.