DNA repair is a key cellular process related to disease prevention and genome correction that occurs within the crowded environment of the cell nucleus. Surprisingly, the majority of DNA damage search and repair studies have been carried out in dilute solution and little detail is known about the effects of solution crowding, despite the fact that up to 40% of the cellular volume is occupied by biomolecules. 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 a profound effect on the kinetic properties of DNA repair enzymes. Here, we have used a continuous fluorescence assay to determine the effects of inert polymers (“crowders”) on the steady-state kinetic parameters of uracil excision by human uracil DNA glycosylase (hUNG). Increasing the concentration of polyethylene glycol (PEG) 8000 [0-20% (w/v)] was found to reduce both Km and kcat, resulting in a negligible effect on kcat/Km. A comparison of the effect of PEG on diffusion controlled kcat/Km to what is expected based solely on solution viscosity dependent changes in the hUNG diffusion coefficient (Stokes-Einstein relation) revealed that viscosity alone is not sufficient to account for the trend observed. We also show that these effects cannot be attributed to the minimal changes in ion activity in the presence of PEG. We suggest that the excluded volume effect induced by the presence of molecular crowding agents facilitates protein-DNA complex formation and limits product release, providing a means by which the cellular environment could greatly impact DNA damage recognition.

Compensatory kinetic effects of viscogen and crowding agent PEG8000 on the reaction of human uracil DNA glycosylase (hUNG)

249th National Meeting (2015)

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