Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation

Abstract

Nuclear human uracil–DNA glycosylase (hUNG2) initiates base excision repair (BER) of genomic uracils generated through misincorporation of dUMP or through deamination of cytosines. Like many human DNA glycosylases, hUNG2 contains an unstructured N–terminal domain that encodes a nuclear localization signal, protein binding motifs, and sites for post–translational modifications. Although the N–terminal domain has minimal effects on DNA binding and uracil excision kinetics, we report that this domain enhances the ability of hUNG2 to translocate on DNA chains as compared to the catalytic domain alone. The enhancement is most pronounced when physiological ion concentrations and macromolecular crowding agents are used. These data suggest that crowded conditions in the human cell nucleus promote the interaction of the N–terminus with duplex DNA during translocation. The increased contact time with the DNA chain likely contributes to the ability of hUNG2 to locate densely spaced uracils that arise during somatic hypermutation and during fluoropyrimidine chemotherapy.

N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA–dsDNA junctions

“…In the presence of hUNG2 alone, all of the DNA fragments were approximately equal in intensity, indicating that U21 and U45 were kinetically equivalent under these conditions. The lack of discrimination between uracil sites was anticipated based on previous studies that measured hUNG2 activity on DNA duplexes containing two uracil bases at various spacings ( 16 , 17 , 20 , 26 ).…”

“…This study and previous work strongly suggest that the NTD of hUNG2 interacts with duplex and ssDNA. We previously proposed that the NTD interacted with dsDNA based on the observation that full-length hUNG2 translocated along duplex DNA more efficiently than the isolated catalytic domain ( 17 ). Results from the present study are consistent with this proposal because hUNG2 has a slightly higher affinity for duplex DNA than the catalytic domain alone (Figure 4 ).…”

“…The procedures for the expression and purification of full-length hUNG2 ( 10 ) and the hUNG2 catalytic domain (amino acids 92–313) have been described previously ( 16 , 17 ). Heterotrimeric RPA was expressed and purified using the p11d-tRPA plasmid as described previously ( 10 , 18 , 19 ).…”

“…Because the hUNG2 N-terminal domain bound very weakly in this buffer, we also performed experiments at a lower NaCl concentration of 20 mM. Individual solutions of protein and DNA (150 μl) were equilibrated for at least 5 min before measuring the fluorescence anisotropy ( 16 , 17 ). The anisotropy values were plotted against total protein concentration and fit to a quadratic binding isotherm as described previously ( 10 , 17 , 22 ).…”

Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu

“…Recent experiments (26)(27)(28), however, suggest faster diffusion and enhanced enzyme activity of DNA binding proteins in the presence of molecular crowders. For example, in vitro studies on the hydrolysis of DNA by endonucleases DNase I and S1 nuclease increase substantially in a medium crowded by polyethylene glycol (PEG) (29).…”