Fidelity of uracil-initiated base excision DNA repair in DNA polymerase beta-proficient and -deficient mouse embryonic fibroblast cell extracts.

Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase beta (+/+) and (-/-) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U.A, U.G, and U.T targets contained within the lacZalpha gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase beta (+/+) and (-/-) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced ( approximately 88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting approximately 20% of the repair events. Addition of p21 peptide or protein to DNA polymerase beta (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by approximately 2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U.T) DNA was determined to be 5.7-7.2 x 10(-4) when using DNA polymerase beta (+/+) and (-/-) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U.G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U.G target and frequently involved a 1-base deletion or incorporation of dTMP.

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.

Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts.

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung(+) dug(+)), approximately 80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung(-) dug(+)), BH157 (ung(+) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be approximately 5.5 x 10(-)(4) with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.

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.

Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV.

Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption-ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U.G, U.A, ethenoC.G, and ethenoC.A targets; however, activity was not detected on DNA containing a T.G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U.G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP. G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP.G target, while binding was not observed on a substrate incised 5' to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U.G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.

Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis.

The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated. Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung. The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues. Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0. Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer. However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate. Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil. Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP). Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D [Putnam, C. D., Shroyer, M. J. N., Lundquist, A. J., Mol, C. D., Arvai, A. S., Mosbaugh, D. W., and Tainer, J. A. (1999) J. Mol. Biol. 287, 331-346] provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage. A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.

Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi). A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity. High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi. The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes. Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA. Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex. Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands. A similar beta-zipper is observed in the conversion between the open and closed forms of UDG. The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity.

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 lacZalpha 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 lacZalpha 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.

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 alpha2-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.

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.

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 beta-strands and two alpha-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 beta-strands. This negatively charged region is likely to play an important role in the binding of Ugi to uracil-DNA glycosylase.

Processivity of Escherichia coli and rat liver mitochondrial uracil-DNA glycosylase is affected by NaCl concentration.

Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U.G mispair was approximately 2-fold greater than a U.A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end 32P-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U.G or U.A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [32P]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [32P]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U.A- and U.G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (> or = 50 mM) caused reduced processivity on both U.A- and U.G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.

Secondary 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 (Wang, Z., and Mosbaugh, D. W. (1989) J. Biol. Chem. 264, 1163-1171). The secondary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance. The protein adopts a single well defined structure consisting of five anti-parallel beta-strands and two alpha-helices. Six loop or turn regions were identified that contain approximately one half of the acidic amino acid residues and connect the beta-strands sequentially to one another. The secondary structure suggests which regions of Ugi may be involved in interactions with uracil-DNA glycosylase.

UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA. Identification of amino acid residues in the single-stranded DNA binding site.

Photochemical cross-linking of Escherichia coli uracil-DNA glycosylase (Ung) to oligonucleotide dT20 was performed to identify amino acid residues that reside in or near the DNA-binding site. UV-catalyzed cross-linking reactions produced a covalent Ung x dT20 complex which was resolved from uncross-linked enzyme by SDS-polyacrylamide gel electrophoresis. Cross-link formation required native Ung and was inhibited by increasing concentrations of NaCl in a manner characteristics of NaCl inhibition of Ung catalytic activity. The Ung x dT20 complex was purified to apparent homogeneity, and mass spectrometry revealed that Ung was cross-linked to dT20 in 1:1 stoichiometry as a 31,477 dalton complex. Purified Ung x dT20 lacked detectable uracil-DNA glycosylase activity and failed to bind single-stranded DNA. Recently, we demonstrated that the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) binds Ung and prevents further interaction with DNA (Bennett, S. E., Schimerlik, M. I., and Mosbaugh, D. W. (1993) J. Biol. Chem. 268, 26879-26885). Addition of the Ugi protein to the cross-linking reaction blocked formation of the Ung x dT20 cross-link. Conversely, the Ung x dT20 cross-link was refractory to Ugi binding. Upon trypsin digestion of Ung x dT20, four distinct products were identified as peptide x dT20 cross-links. A combination of amino acid sequence and mass spectrometric analysis revealed that four tryptic peptides (T6, T18, T19, and T18/19) were adducted to dT20. These observations suggest that dT20 is cross-linked to the Ung DNA-binding site.

Kinetics of the uracil-DNA glycosylase/inhibitor protein association. Ung interaction with Ugi, nucleic acids, and uracil compounds.

The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) inactivates Escherichia coli uracil-DNA glycosylase (Ung) by forming an Ung.Ugi protein complex with 1:1 stoichiometry. Stability of the Ung.Ugi complex was demonstrated by the inability of free Ugi to exchange with Ugi bound in preformed complex. Ung was reacted with fluorescein 5-isothiocyanate to produce fluorescent-Ung (F-Ung), which retained full uracil-DNA glycosylase activity and susceptibility to Ugi inactivation. Addition of Ugi to F-Ung under steady-state conditions resulted in saturable (15%) fluorescence quenching at a F-Ung.Ugi ratio of 1:1.4. Dissociation constants determined for the F-Ung interaction with M13 DNA, uracil-containing DNA, and poly(U) equaled 600, 220, and 190 microM, respectively. While F-Ung associated with nucleic acid polymers was able to bind Ugi efficiently, F-Ung bound in the F-Ung.Ugi complex could no longer effectively bind nucleic acid. Stopped-flow kinetic analysis suggested the F-Ung/Ugi association was described by a two-step mechanism. The first step entailed a rapid pre-equilibrium distinguished by the dissociation constant Kd = 1.3 microM. The second step led irreversibly to the formation of the final complex and was characterized by the rate constant k = 195 s-1. We infer Ugi inactivates Ung through the formation of an exceptionally stable protein-protein complex.

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.

Characterization of the Escherichia coli uracil-DNA glycosylase.inhibitor protein complex.

The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein was characterized and shown to form a stable complex with Escherichia coli uracil-DNA glycosylase (Ung). As determined by mass spectrometry, the Ugi protein had a molecular weight of 9,474. We confirmed this value by sedimentation equilibrium centrifugation and determined that Ugi exists as a monomeric protein in solution. Amino acid analysis performed on both Ugi and Ung proteins was in excellent agreement with the amino acid composition predicted from the respective nucleotide sequence of each gene. The Ung.Ugi complex was resolved from its constitutive components by nondenaturing polyacrylamide gel electrophoresis and shown to possess a 1:1 stoichiometry. Analytical ultracentrifugation studies revealed that the Ung.Ugi complex had a molecular weight of 35,400, consistent with the complex containing one molecule each of Ung and Ugi. The acidic isoelectric points of the protein species were 6.6 (Ung) and 4.2 (Ugi), whereas the Ung.Ugi complex had an isoelectric point of 4.9. Dissociation of the Ung.Ugi complex by SDS-polyacrylamide gel electrophoresis revealed no apparent alteration in the molecular weight of either polypeptide subsequent to binding. Furthermore, when the Ung.Ugi complex was treated with urea and resolved by urea-polyacrylamide gel electrophoresis, both uracil-DNA glycosylase and inhibitor activities were recovered from the dissociated complex. Thus, the complex seems to be reversible. In addition, we demonstrated that the Ugi interaction with Ung prevents enzyme binding to DNA and dissociates uracil-DNA glycosylase from a preformed DNA complex.

Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision.

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.