Dale W. Mosbaugh

Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: Protein mimicry of DNA

Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.

Uracil-Excision DNA Repair

Publisher Summary Uracil residues are introduced into prokaryotic and eukaryotic deoxyribonucleic acid (DNA) as a normal physiological process during DNA synthesis, and by spontaneous chemical modification of cytosine residues in DNA; thus, the acquisition of uracil in cellular DNA is unavoidable. However, the rate of uracil accumulation may vary significantly, depending on the ratio of deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) in intracellular pools and on whether the cells are exposed to cytosinedeaminating agents. The biological consequences of uracil residues in DNA may have cytotoxic, mutagenic, or lethal effects. An uncontrolled accumulation of the uracil residues in DNA leads to various perturbations of molecular events, ranging from altered protein-nucleic acid interactions to uracil-DNA degradation. The importance of eliminating uracil from DNA is underscored, by the observation that the uracil-DNA repair pathway of almost every organism examined, is remarkably similar. It appears that not only is one nucleotide DNA repair evident in E. coli as well as in human cells, but also that uracil-DNA glycosylase is one of the most highly conserved polypeptides yet identified.

Fidelity and Mutational Specificity of Uracil-initiated Base Excision DNA Repair Synthesis in Human Glioblastoma Cell Extracts

The fidelity of DNA synthesis associated with uracil-initiated base excision repair was measured in human whole cell extracts. An M13mp2 lacZα DNA-based reversion assay was developed to assess the error frequency of DNA repair synthesis at a site-specific uracil residue. All three possible base substitution errors were detected at the uracil target causing reversion of opal codon 14 in the Escherichia coli lacZα gene. Using human glioblastoma U251 whole cell extracts, approximately 50% of the heteroduplex uracil-containing DNA substrate was completely repaired, as determined by the insensitivity of form I DNA reaction products to cleavage by a combined treatment of E. coli uracil-DNA glycosylase and endonuclease IV. The majority of repair occurred by the uracil-initiated base excision repair pathway, since the addition of the bacteriophage PBS2 uracil-DNA glycosylase inhibitor protein to extracts significantly blocked this process. In addition, the formation of repaired form I DNA molecules occurred concurrently with limited DNA synthesis, which was largely restricted to the HinfI DNA fragment initially containing the uracil residue and specific to the uracil-containing DNA strand. Based on the reversion frequency of repaired M13mp2 DNA, the fidelity of DNA repair synthesis at the target was determined to be about one misincorporated nucleotide per 1900 repaired uracil residues. The major class of base substitutions propagated transversion mutations, which were distributed almost equally between T to G and T to A changes in the template. A similar mutation frequency was also observed using whole cell extracts from human colon adenocarcinoma LoVo cells, suggesting that mismatch repair did not interfere with the fidelity measurements.

Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2.

A plasmid expression vector (pZWtac1) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene (ugi) in Escherichia coli. In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter. Constitutive expression of the ugi was observed in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG). In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx. 16-fold higher level, and accounted for approx. 19% of the total soluble cellular proteins. Following high-level production in E. coli, the Ugi protein was purified to apparent homogeneity. Using E. coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner. Kinetic studies revealed a Ki value (0.14 microM) of approx. twelve-fold lower than Km value (1.7 microM) of glycosylase. Ugi did not act synergistically with free uracil to inhibit E. coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.

Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase

The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophage PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight enzyme:inhibitor complex. To create human cells that are impaired for UDG activity, the human glioma U251 cell line was engineered to produce active Ugi protein. In vitro assays of crude cell extracts from several Ugi-expressing clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell lines were characterized for their ability to conduct in vivo uracil-DNA repair. Whereas transfected plasmid DNA containing either a U:G mispair or U:A base pairs was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi. Experiments using a shuttle vector to detect mutations in a target gene showed that Ugi-expressing cells exhibited a 3-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions. The growth rate and cell cycle distribution of Ugi-expressing cells did not differ appreciably from their parental cell counterpart. Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate Ugi-insensitive excision of uracil bases from DNA was not detected in the parental U251 cells. However, a Ugi-insensitive UDG activity of unknown origin that recognizes U:G mispairs and to a lesser extent U:A base pairs in duplex DNA, but which was inactive toward uracil residues in single-stranded DNA, was detected under assay conditions previously shown to be efficient for detecting TDG.

Site-directed Mutagenesis and Characterization of Uracil-DNA Glycosylase Inhibitor Protein ROLE OF SPECIFIC CARBOXYLIC AMINO ACIDS IN COMPLEX FORMATION WITH ESCHERICHIA COLI URACIL-DNA GLYCOSYLASE

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex. Seven mutant Ugi proteins (E20I, E27A, E28L, E30L, E31L, D61G, and E78V) were created to assess the role of various negatively charged residues in the binding mechanism. Each mutant Ugi protein was purified and characterized with respect to inhibitor activity and Ung binding properties relative to the wild type Ugi. Analysis of the Ugi protein solution structures by nuclear magnetic resonance indicated that the mutant Ugi proteins were folded into the same general conformation as wild type Ugi. All seven of the Ugi proteins were capable of forming a Ung·Ugi complex but varied considerably in their individual ability to inhibit Ung activity. Like the wild type Ugi, five of the mutants formed an irreversible complex with Ung; however, the binding of Ugi E20I and E28L to Ung was shown to be reversible. The tertiary structure of [13C,15N]Ugi in complex with Ung was determined by solution state multi-dimensional nuclear magnetic resonance and compared with the unbound Ugi structure. Structural and functional analysis of these proteins have elucidated the two-step mechanism involved in Ung·Ugi association and irreversible complex formation.

IDENTIFICATION OF SPECIFIC CARBOXYL GROUPS ON URACIL-DNA GLYCOSYLASE INHIBITOR PROTEIN THAT ARE REQUIRED FOR ACTIVITY

The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by forming an exceptionally stable protein-protein complex in which Ugi mimics electronegative and structural features of duplex DNA (Beger, R. D., Balasubramanian, S., Bennett, S. E., Mosbaugh, D. W., and Bolton, P. H. (1995) J. Biol. Chem. 270, 16840-16847; Mol, C. D., Arvai, A. S., Sanderson, R. J., Slupphaug, G., Kavli, B., Krokan, H. E., Mosbaugh, D. W., and Tainer, J. A. (1995) Cell 82, 701-708). The role of specific carboxylic amino acid residues in forming the Ung·Ugi complex was investigated using selective chemical modification techniques. Ugi treated with carbodiimide and glycine ethyl ester produced five discrete protein species (forms I-V) that were purified and characterized. Analysis by mass spectrometry revealed that Ugi form I escaped protein modification, and forms II-V showed increasing incremental amounts of acyl-glycine ethyl ester adduction. Ugi forms II-V retained their ability to form a Ung·Ugi complex but exhibited a reduced ability to inactivate Escherichia coli Ung, directly reflecting the extent of modification. Competition experiments using modified forms II-V with unmodified Ugi as a competitor protein revealed that unmodified Ugi preferentially formed complex. Furthermore, unmodified Ugi and poly(U) were capable of displacing forms II-V from a preformed Ung·Ugi complex but were unable to displace Ugi form I. The primary sites of acyl-glycine ethyl ester adduction were located in the α2-helix of Ugi at Glu-28 and Glu-31. We infer that these two negatively charged amino acids play an important role in mediating a conformational change in Ugi that precipitates the essentially irreversible Ung/Ugi interaction.

Tertiary Structure of Uracil-DNA Glycosylase Inhibitor Protein

The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that inactivates uracil-DNA glycosylase from diverse organisms. The secondary structure of Ugi consists of five anti-parallel β-strands and two α-helices (Balasubramanian, S., Beger, R. D., Bennett, S. E., Mosbaugh, D. W., and Bolton, P. H.(1995) J. Biol. Chem. 270, 296-303). The tertiary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance. The Ugi structure contains an area of highly negative electrostatic potential produced by the close proximity of a number of acidic residues. The unfavorable interactions between these acidic residues are apparently accommodated by the stability of the β-strands. This negatively charged region is likely to play an important role in the binding of Ugi to uracil-DNA glycosylase.

In situ detection of DNA-metabolizing enzymes following polyacrylamide gel electrophoresis.

We have presented several protocols for producing an in situ activity gel that allows detection of various DNA-metabolizing enzymes. Both nondenaturing polyacrylamide and SDS-polyacrylamide activity gel electrophoresis procedures were detailed. Combining the use of defined [32P]DNA substrates with product analysis, these procedures detected a wide spectrum of enzymatic activities. The ability to detect 7 different catalytic activities of 15 different enzymes provides encouragement for expanded applications. It is hoped that others will find this technique applicable for detecting these enzymes and other activities in different biological systems. The modification of DNA in situ and the creation of intermediate substrates within activity gels should prove extremely useful for dissecting the enzymatic steps of DNA replication, repair, recombination, and restriction, as well as the metabolic pathways of other nucleic acids.

Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts.

The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 lacZ alpha DNA-based reversion assay. Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZ alpha gene. Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E. coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred. Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides. The misincorporation frequency of BER DNA synthesis at the target site was 5.2 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to G > T to A >> T to C. Uracil-initiated BER DNA synthesis in extracts of E. coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined. Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 x 10(-4). A reduced, but detectable level of BER was observed in extracts of E. coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold.