@article{1299940,

title = {Investigation of N-Terminal Phospho-Regulation of~Uracil DNA Glycosylase Using Protein Semisynthesis},

author = {Brian P Weiser and James T Stivers and Philip A Cole},

doi = {10.1016/j.bpj.2017.06.016},

issn = {1542-0086},

year  = {2017},

date = {2017-07-01},

journal = {Biophys J},

volume = {113},

number = {2},

pages = {393-401},

abstract = {Uracil DNA Glycosylase (UNG2) is the primary enzyme in humans that prevents the stable incorporation of deoxyuridine monophosphate into DNA in the form of U/A basepairs. During S-phase, UNG2 remains associated with the replication fork through its interactions with two proteins, Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA), which are critical for DNA replication and repair. In this work, we used protein semisynthesis and fluorescence anisotropy assays to explore the interactions of UNG2 with PCNA and RPA and to determine the effects of two UNG2 phosphorylation sites (Thr6 and Tyr8) located within its PCNA-interacting motif (PIP-box). In binding assays, we found that phosphorylation of Thr6 or Tyr8 on UNG2 can impede PCNA binding without affecting UNG2 catalytic activity or its RPA interaction. Our data also suggests that unmodified UNG2, PCNA, and RPA can form a ternary protein complex. We propose that the UNG2 N-terminus may serve as a flexible scaffold to tether PCNA and RPA at the replication fork, and that post-translational modifications on the UNG2 N-terminus disrupt formation of the PCNA-UNG2-RPA protein complex.},

keywords = {Catalysis, DNA Glycosylases, Electrospray Ionization, Escherichia coli, Humans, Mass, Mutation, Phosphorylation, Proliferating Cell Nuclear Antigen, Protein Binding, Protein Domains, Protein Stability, Replication Protein A, Spectrometry},

pubstate = {published},

tppubtype = {article}

}

@article{1299944,

title = {A Tunable Brake for HECT Ubiquitin Ligases},

author = {Zan Chen and Hanjie Jiang and Wei Xu and Xiaoguang Li and Daniel R Dempsey and Xiangbin Zhang and Peter Devreotes and Cynthia Wolberger and L Mario Amzel and Sandra B Gabelli and Philip A Cole},

doi = {10.1016/j.molcel.2017.03.020},

issn = {1097-4164},

year  = {2017},

date = {2017-05-01},

journal = {Mol Cell},

volume = {66},

number = {3},

pages = {345-357.e6},

abstract = {The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules, and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study, we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analyses have revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior.},

keywords = {Allosteric Regulation, Endosomal Sorting Complexes Required for Transport, Enzyme Activation, Enzyme Stability, HeLa Cells, Humans, Models, Molecular, Mutation, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Domains, Protein Processing, Proteolysis, Repressor Proteins, Structure-Activity Relationship, Transfection, Ubiquitin-Protein Ligases},

pubstate = {published},

tppubtype = {article}

}

@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}

}