Molecular Crowding Enhances Facilitated Diffusion of Two Human DNA Glycosylases

The cell nucleus is a complex environment in which approximately 40% of the nuclear volume is occupied by a variety of macromolecules that greatly affect solution viscosity and the amount of available intracellular space. An expected consequence of macromolecular crowding is an entropic “depletion” effect that favors the formation of compact complexes over free components that consume a larger volume. This could have profound consequences for the kinetic, thermodynamic and structural properties of enzymes. Artificial crowding agents, such as PEG polymers, have historically been used to model the effects of macromolecular crowding on enzyme reactions and protein-protein association. Here we report the effects of PEG polymers on the ability of human uracil (hUNG) and 8-oxoguanine DNA glycosylases (hOGG1) to execute intramolecular transfer between damaged sites within the context of duplex DNA. Although molecular crowding resulted in only a small decrease in DNA binding affinity, intramolecular translocation was dramatically enhanced. The mechanism involves the consequences of microenvironments of low viscosity generated by “cages” formed around the protein and DNA by large crowding agents. Dissociative transfers involving short-range dissociation and rebinding to DNA (“hopping”) within the low-viscosity cage appeared to be unaffected while the efficiency and distance of associative transfers, where the enzyme remains in close contact with the DNA chain (“sliding”), are greatly increased. We show that this has a significant impact on enzymatic turnover of hUNG and provide evidence of association enhancement and reduced likelihood of complex dissociation once the enzyme is bound to DNA. We suggest that crowded environments favor the compact enzyme-DNA complex, which provides a mechanism by which the cellular environment could play a major role in facilitating DNA damage recognition in human cells.