2017
Esadze, Alexandre; Rodriguez, Gaddiel; Weiser, Brian P; Cole, Philip A; Stivers, James T
Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells Journal Article
In: Nucleic Acids Res, vol. 45, no. 21, pp. 12413-12424, 2017, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: Cell Line, DNA, DNA Glycosylases, Humans, Uracil
@article{1299936,
title = {Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells},
author = {Alexandre Esadze and Gaddiel Rodriguez and Brian P Weiser and Philip A Cole and James T Stivers},
doi = {10.1093/nar/gkx848},
issn = {1362-4962},
year = {2017},
date = {2017-12-01},
journal = {Nucleic Acids Res},
volume = {45},
number = {21},
pages = {12413-12424},
abstract = {DNA ’sliding’ by human repair enzymes is considered to be important for DNA damage detection. Here, we transfected uracil-containing DNA duplexes into human cells and measured the probability that nuclear human uracil DNA glycosylase (hUNG2) excised two uracil lesions spaced 10-80 bp apart in a single encounter without escaping the micro-volume containing the target sites. The two-site transfer probabilities were 100% and 54% at a 10 and 40 bp spacing, but dropped to only 10% at 80 bp. Enzyme trapping experiments suggested that site transfers over 40 bp followed a DNA ’hopping’ pathway in human cells, indicating that authentic sliding does not occur even over this short distance. The transfer probabilities were much greater than observed in aqueous buffers, but similar to in vitro measurements in the presence of polymer crowding agents. The findings reveal a new role for the crowded nuclear environment in facilitating DNA damage detection.},
keywords = {Cell Line, DNA, DNA Glycosylases, Humans, Uracil},
pubstate = {published},
tppubtype = {article}
}
DNA ’sliding’ by human repair enzymes is considered to be important for DNA damage detection. Here, we transfected uracil-containing DNA duplexes into human cells and measured the probability that nuclear human uracil DNA glycosylase (hUNG2) excised two uracil lesions spaced 10-80 bp apart in a single encounter without escaping the micro-volume containing the target sites. The two-site transfer probabilities were 100% and 54% at a 10 and 40 bp spacing, but dropped to only 10% at 80 bp. Enzyme trapping experiments suggested that site transfers over 40 bp followed a DNA ’hopping’ pathway in human cells, indicating that authentic sliding does not occur even over this short distance. The transfer probabilities were much greater than observed in aqueous buffers, but similar to in vitro measurements in the presence of polymer crowding agents. The findings reveal a new role for the crowded nuclear environment in facilitating DNA damage detection.
0000
Rodriguez, Gaddiel; Esadze, Alexandre; Weiser, Brian P; Schonhoft, Joseph D; Cole, Philip A; Stivers, James T
Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation Journal Article
In: ACS Chem Biol, vol. 12, no. 9, pp. 2260-2263, 0000, ISSN: 1554-8937.
Abstract | Links | BibTeX | Tags: Binding Sites, Biological Transport, DNA, DNA Glycosylases, Humans, Nuclear Localization Signals, Protein Domains
@article{1299939,
title = {Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation},
author = {Gaddiel Rodriguez and Alexandre Esadze and Brian P Weiser and Joseph D Schonhoft and Philip A Cole and James T Stivers},
doi = {10.1021/acschembio.7b00521},
issn = {1554-8937},
journal = {ACS Chem Biol},
volume = {12},
number = {9},
pages = {2260-2263},
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.},
keywords = {Binding Sites, Biological Transport, DNA, DNA Glycosylases, Humans, Nuclear Localization Signals, Protein Domains},
pubstate = {published},
tppubtype = {article}
}
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.