2017
Weiser, Brian P; Stivers, James T; Cole, Philip A
Investigation of N-Terminal Phospho-Regulation of~Uracil DNA Glycosylase Using Protein Semisynthesis Journal Article
In: Biophys J, vol. 113, no. 2, pp. 393-401, 2017, ISSN: 1542-0086.
Abstract | Links | BibTeX | Tags: 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
@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}
}
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.
2014
Wang, Yun; Kavran, Jennifer M; Chen, Zan; Karukurichi, Kannan R; Leahy, Daniel J; Cole, Philip A
Regulation of S-adenosylhomocysteine hydrolase by lysine acetylation Journal Article
In: J Biol Chem, vol. 289, no. 45, pp. 31361-72, 2014, ISSN: 1083-351X.
Abstract | Links | BibTeX | Tags: Acetylation, Adenosylhomocysteinase, Amino Acid, Amino Acid Sequence, Catalysis, Crystallography, Humans, Hydrogen Bonding, Lysine, Methylation, Models, Molecular, Molecular Sequence Data, Mutagenesis, NAD, Plasmids, Post-Translational, Protein Binding, Protein Processing, Protein Structure, Recombinant Proteins, Sequence Homology, Site-Directed, Structure-Activity Relationship, Tertiary, X-Ray
@article{1299977,
title = {Regulation of S-adenosylhomocysteine hydrolase by lysine acetylation},
author = {Yun Wang and Jennifer M Kavran and Zan Chen and Kannan R Karukurichi and Daniel J Leahy and Philip A Cole},
doi = {10.1074/jbc.M114.597153},
issn = {1083-351X},
year = {2014},
date = {2014-11-01},
journal = {J Biol Chem},
volume = {289},
number = {45},
pages = {31361-72},
abstract = {S-Adenosylhomocysteine hydrolase (SAHH) is an NAD(+)-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys(401) and Lys(408) but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys(401) and Lys(408) and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD(+) binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.},
keywords = {Acetylation, Adenosylhomocysteinase, Amino Acid, Amino Acid Sequence, Catalysis, Crystallography, Humans, Hydrogen Bonding, Lysine, Methylation, Models, Molecular, Molecular Sequence Data, Mutagenesis, NAD, Plasmids, Post-Translational, Protein Binding, Protein Processing, Protein Structure, Recombinant Proteins, Sequence Homology, Site-Directed, Structure-Activity Relationship, Tertiary, X-Ray},
pubstate = {published},
tppubtype = {article}
}
S-Adenosylhomocysteine hydrolase (SAHH) is an NAD(+)-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys(401) and Lys(408) but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys(401) and Lys(408) and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD(+) binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.