Thermodynamic and Kinetic Constants of Incorporation in a DNA Polymerase Measured at the Single Molecule Level: Cytosine versus Modified Cytosines

Abstract

Conventional analysis of biological systems is limited in part, because it provides ensemble-averaged information. Such limitation can be particularly problematic when variation among single molecules exists. Atomic force microscopy (AFM) is one of the well-established methods for unveiling molecular behavior at the single-molecule level. Since the inception of AFM, the tool has been employed to investigate key information in the life sciences at the single-molecule level, such as folding and unfolding of proteins, recognition, thermodynamics, and the kinetics of molecular interactions. It is possible to measure pico-Newton force under physiological conditions, which provide molecular energy landscape and kinetic information through single-molecule recognition. Also, the sub-nanometer lateral resolution of AFM has been widely used for the spatial distribution mapping of specific biomolecules and localizing individual recognition sites on various surfaces. To enhance the precision and reproducibility of the interaction force, it is important to adopt a well-controlled surface for the AFM probe. Employment of such surfaces for target substrates will upgrade the reliability of outcomes further. We have observed that the dendron-coated surface is one of the kinds meeting such goal, and the probability of collecting truly 1:1 interaction increases noticeably, we took such an approach in this study.

Incorporation of Cytosine and Modified Cytosines in Human DNA Polymerase Delta 1: Thermodynamic and Kinetic Constants at the Single Molecule Level DNA methylation plays key roles in gene expression, regulation, epigenetics, and cancer in various areas. A lot of efforts have been made to understand the methylation, and studies with methyl binding proteins, mutated proteins, and chemical structure modification of nucleotides are examples. DNA methylation process typically converts cytosine (C) to 5-methylcytosine (5mC) in eukaryotes, and it is important to understand the process by which DNA is replicated through DNA polymerases associated with the methylated DNA. Because replication is one of the important life phenomena in which genetic information is preserved, characterizing the fundamental constants such as binding kinetics and thermodynamics of complementary nucleotides to the enzymatic pocket could help to understand the replication process. Additionally, the effect of 5-carboxycytosine (5caC), a form produces by iterative oxidation of 5mC, on the DNA replication process is still not well known yet. Therefore, it is useful to retrieve such fundamental constants at the single-molecule associated with 5mC and 5caC. The human DNA polymerase delta 1 (p125) was immobilized at the tip of an atomic force microscope (AFM) with orientation control, and interaction between the deoxyguanosine triphosphate (dGTP) immobilized on a solid substrate and p125 in the presence of a DNA duplex was observed. We controlled the orientation of the DNA polymerases (DNAP) so that it could interact without hindrance, and by increasing the contact time, we could increase the binding probability. It was observed that the interaction probability increased with the increasing concentration of DNA strands, and by fitting the change, the equilibrium constant between DNAP and DNA was derived. Increasing the concentration of dGTP in the reaction solution decreased the interaction probability and through the fitting, we were able to derive the equilibrium constant between dGTP and DNAP that retains the DNA template in the binding pocket. Affinity and kinetic constants for cytosine (C), 5mC, and 5caC in the DNAP were compared in terms of the steric and electronic effect of the substituents at the 5-position of cytosine as the dissociation constant could be obtained through the loading rate dependence of the specific unbinding force value.