The Role of Leucine 191 of Escherichia coli Uracil DNA Glycosylase in the Formation of a Highly Stable Complex with the Substrate Mimic, Ugi, and in Uracil Excision from the Synthetic Substrates*

Priya HandaDagger, Sudipta Roy, and Umesh Varshney§

From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India

Received for publication, December 12, 2000, and in revised form, February 6, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, initiates the uracil excision repair pathway. Ugi, a bacteriophage-encoded peptide, potently inhibits UDGs by serving as a remarkable substrate mimic. Structure determination of UDGs has identified regions important for the exquisite specificity in the detection and removal of uracils from DNA and in their interaction with Ugi. In this study, we carried out mutational analysis of the Escherichia coli UDG at Leu191 within the 187HPSPLS192 motif (DNA intercalation loop). We show that with the decrease in side chain length at position 191, the stability of the UDG-Ugi complexes regresses. Further, while the L191V and L191F mutants were as efficient as the wild type protein, the L191A and L191G mutants retained only 10 and 1% of the enzymatic activity, respectively. Importantly, however, substitution of Leu191 with smaller side chains had no effect on the relative efficiencies of uracil excision from the single-stranded and a corresponding double-stranded substrate. Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of UDG, and it contributes to the formation of a physiologically irreversible complex with Ugi. We also envisage a role for Leu191 in stabilizing the productive enzyme-substrate complex.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DNA in cells is unceasingly subjected to damages that occur under normal physiological conditions and can be exacerbated by environmental mutagens. If left unrepaired, these can be detrimental to the maintenance of genetic integrity (1). Uracil is a natural component of RNA but can occur in the DNA by spontaneous deamination of an inherently unstable base, cytosine (2). Occasional incorporation of dUMP, in place of dTMP, during DNA synthesis is yet another way by which uracil can arise in DNA (3). If left unrepaired, in a U:G mismatch, uracil is promutagenic and can lead to GCright-arrowAT transition mutations during the next round of replication. Further, uracil, in an A:U pair in the DNA sequences can impend their recognition by the cognate regulatory proteins (4). In cells, a highly efficient base excision repair enzyme, uracil-DNA glycosylase (UDG),1 is dedicated to the indomitable task of freeing the DNA from uracil residues (5, 6). UDGs have been identified from a large number of prokaryotic and eukaryotic organisms, and these enzymes show a high degree of conservation from bacteria to viruses to humans (7, 8). UDGs also interact with a number of proteins such as the Bacillus subtilis phage-encoded uracil DNA glycosylase inhibitor, Ugi, and host cellular factors such as single-stranded DNA-binding protein and proliferating cell nuclear antigen (9-12). Thus, UDGs constitute a remarkably interesting model system to understand the basis of catalytic prowess and specificity associated with protein-DNA and protein-protein interactions. The mechanism of uracil excision that has emerged from various structural studies and mutational analyses of UDGs is that the glycosidic bond between uracil and the sugar is cleaved by the attack of a hydroxyl nucleophile on the deoxyribose C1' atom. This nucleophile is generated by the activation of a water molecule by an absolutely conserved Asp of the GQDPYH motif. Concomitant protonation of the O2 of the uracil base by His of yet another highly conserved motif, HPSPLS, enhances its leaving group quality (13, 14).

The crystal structures of Escherichia coli UDG (15-17) reveal that there is a remarkable conservation of the overall architecture and the active site geometry between the UDGs from human, bacterial, and viral sources. However, several crucial residues (Gln63, Asp64, and Leu191) in E. coli UDG show subtle conformational differences when compared with those in human UDG (15, 16). The cocrystal structures of the human herpes simplex virus-1 and E. coli UDGs with Ugi (15, 17-19) reveal that Ugi binds UDG at the active site face and provides one of the most outstanding examples of molecular mimicry of the DNA substrate.

The major interactions, which render the complex between UDG and Ugi physiologically irreversible, are defined by (i) hydrogen bonding and packing contacts derived from the complementarity between the conserved Leu loop (187HPSPLS192) of E. coli UDG and eight hydrophobic residues of Ugi (Met24, Val29, Val32, Ile33, Val43, Met56, Leu58, and Val71) in a cavity burrowed between the alpha 2 and the antiparallel beta  sheet of Ugi and (ii) the electrostatic interactions between the acidic residues of the beta 1 edge of Ugi with the key active site residues of E. coli UDG. This structure also showed that the interaction between UDG and Ugi results in the burial of about 2200 Å2 of the total accessible surface area. The nestling of Leu191 into the hydrophobic cavity of Ugi alone causes the exclusion of about 250 Å2 of the surface area (17).

Mutational and biochemical analyses form a necessary component of understanding the mechanism underlying the macromolecular interactions. However, the only mutational analysis that has been carried out to understand the mechanism of UDG-Ugi interaction has been with respect to the seven acidic residues (Glu20, Glu27, Glu28, Glu30, Glu31, Asp61, and Glu78) of Ugi. With the exception of the E20I and E20L mutants, which formed reversible complexes, the other mutants formed irreversible complexes with E. coli UDG (20). The mutational analysis of neither UDG nor Ugi residues that are involved in the hydrophobic interactions has been performed.

In this study, we have mutated the Leu191 of E. coli UDG to side chains of varying lengths and investigated the effect of these mutations on the ability of the resultant UDGs to form complexes with Ugi under in vitro and in vivo conditions. In addition, we utilized two of these mutants to understand the role of Leu191 in uracil excision from the single- and double-stranded substrates. Importantly, during these studies, we have developed a novel urea-polyacrylamide gel system, which has allowed us to monitor the real time dissociation of UDG-Ugi complexes.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oligodeoxyribonucleotides-- Oligonucleotides were obtained from Bangalore Genei or Ransom Hill Bioscience. The oligomer, SSU9, 5'-d(ctcaagtgUaggcatgcaagagct)-3', is a single-stranded 24-mer oligonucleotide substrate containing dU at the 9th position (from the 5'-end). The substrate AU9, 5'-d(ctcaagtgUaggcatgcttttgcatgcctacacttga)-3', is a 37-mer hairpin oligonucleotide containing dU in the stem region. In AU9, the dU is in the same sequence context as in SSU9 except that it is located in a double-stranded region.

32P Labeling of Oligodeoxyribonucleotides-- Oligonucleotides (10 pmol) were 5'-32P-end labeled using 10 µCi of [gamma -32P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase in 10-µl reaction volumes (21) and purified by chromatography on Sephadex G-50 minicolumns (22). This procedure routinely resulted in labeling efficiency of ~106 cpm/pmol. For Km and Vmax determinations, radiolabeled oligonucleotides were mixed with cold substrates such that the contribution from labeled substrates was much less than 1% (23).

Generation of the Leu191 E. coli UDG Mutants and Their Overexpression Constructs-- A DNA fragment (~800 base pairs) harboring the sequence downstream of the BamHI site within the ung gene was excised from pTZUng4 using BamHI (24) and subcloned into a similarly digested pTZ19R to generate pTZUng2B'. This recombinant plasmid is similar to pTZUng2B (24) except that it harbors the insert in the opposite orientation. Single-stranded DNA template derived from pTZUng2B' and the mutagenic oligomers 5'-ATG CGC CGA ACC CGG CGA CGG-3', 5'-ATG CGC CGA A (G/C) C CGG CGA CGG-3', and 5'-ATG CGC CGA AAA CGG CGA CGG-3' were used to obtain L191G, L191A, and L191F mutants, respectively (25). All of the mutants were confirmed by complete nucleotide sequencing. During the process of generating these mutations, we serendipitously obtained the L191V mutant. To reconstitute the Leu191 mutants into the pTrcUDG expression vector, the relevant region of the mutant pTZ19R constructs was excised as a NruI-HindIII fragment (~500 base pairs) and subcloned into similarly digested pTrcUDG to generate pTrc99c-based expression constructs for the L191G, L191A, L191V, and L191F mutants. To develop the T7 RNA polymerase-based expression constructs, the ung gene sequences were amplified by polymerase chain reaction from the pTZ19R-based recombinants using Pfu DNA polymerase and a universal primer, 5'-GTTTTCCCAGTCACGAC-3' that anneals to the vector sequence downstream of the multiple cloning site and a gene-specific primer, 5'-CGTGAAGCTTGACGGTACGGGC-3' that anneals in the 3'-flanking region of the ung gene and harbors a HindIII site (shown in italics). After initial heating at 95 °C for 1 min, the reactions were subjected to 24 cycles of temperature shifts at 95 °C for 45 s, 50 °C for 30 s, and 72 °C for 1 min. Finally, the reaction was incubated at 72 °C for 10 min. The polymerase chain reaction products were purified and digested with BamHI and HindIII and used to replace a similar region of the ung gene from a pET11d construct harboring the ung gene open reading frame by using standard recombinant DNA methods (26). The expression constructs in pTrc99c and pET11d thus obtained were reaffirmed by nucleotide sequencing.

Purification of Leu191 UDG Mutants-- The pET11d-based UDG gene constructs were transformed into E. coli BL21 (DE3), and the transformants were inoculated directly into 250 ml of 2YT medium (26). Cells were harvested, and UDG was purified by a modification of the procedure described earlier (6). Briefly, the bacterial cell pellet was suspended in TG buffer (20 mM Tris-HCl, pH 7.4, and 10% (v/v) glycerol) and lysed by sonication. The lysate was clarified by centrifuging at 20,000 × g for 10 min at 4 °C to obtain the S-20 supernatant. Streptomycin sulfate was added to a final concentration of 0.9% to the S-20 extract and subjected to centrifugation at 20,000 × g for 10 min, and the supernatant was subjected to ammonium sulfate fractionation. The pellet from the 40-60% ammonium sulfate saturation was dissolved in TG buffer containing 500 mM NaCl, loaded onto a G-75 column (80 × 7.1 cm2), and eluted with the same buffer. The fractions enriched for UDG were pooled and concentrated by ammonium sulfate fractionation (70% saturation). The precipitate was suspended in 5 ml of HG buffer (20 mM Hepes, pH 7.4, and 10% (v/v) glycerol), dialyzed against the same buffer, and loaded onto a Mono-S column (5 ml; Bio-Rad), which was equilibrated with the HG buffer. The proteins were eluted with a linear gradient of 0-1 M NaCl in HG buffer. The fractions enriched for UDG were dialyzed against TG buffer, loaded onto a HiTrap heparin-Sepharose column (FPLC; Amersham Pharmacia Biotech) equilibrated in the same buffer, and eluted using a gradient of 0-1 M NaCl in TG buffer. The fractions containing apparently pure UDG were pooled, dialyzed against 20 mM Tris-HCl (pH 7.5) and 50% (v/v) glycerol, quantified (27), and stored at -20 °C.

Time Course of Uracil Excision from SSU9-- A reaction mixture (70 µl) was set up in UDG buffer (50 mM Tris-HCl, pH 7.4, 1 mM Na2EDTA, 1 mM dithiothreitol, and 25 µg/ml bovine serum albumin) containing 35 pmol of SSU9. The reaction was started by adding 5 µl of an appropriate dilution of UDG (96.5 pg of wild type, 2250 pg of L191G, 300 pg of L191A, 72 pg of L191V, and 49.5 pg of L191F UDGs) at 37 °C. Aliquots (10 µl) were removed at various time points (0, 2, 4, 6, 8, and 10 min), and the reactions were terminated by adding 5 µl of 0.2 N NaOH and heating at 90 °C for 30 min. The reaction mixture was dried in a SpeedVac (Savant) and taken up in 10 µl of loading dye containing 80% formamide, 0.1% xylene cyanol FF, bromphenol blue, and 1 mM Na2EDTA, and half of the contents was electrophoresed on 15% polyacrylamide-8 M urea gels. The bands corresponding to substrate and product were quantitated by a BioImage Analyser (Fuji).

Determination of Km and Vmax-- Reactions (15 µl) containing varying amounts of 32P-labeled substrates and appropriate concentrations of UDG in the reaction buffer (50 mM Tris-HCl, pH 7.4, 1 mM Na2EDTA, 1 mM dithiothreitol, and 25 µg/ml bovine serum albumin) were incubated at 37 °C for 10 min and stopped by adding 5 µl of 0.2 N NaOH. Cleavage at the abasic site was achieved by heating the contents at 90 °C for 30 min, and the reaction products were analyzed on 15% polyacrylamide-8 M urea gels. The bands corresponding to the product and the remaining substrate were quantified by using a BioImage Analyser (Fuji). Values of Km and Vmax were determined from Hofstee plots (28) of at least two independent experiments.

Formation of UDG-Ugi Complexes in Vitro and Their Analysis on Polyacrylamide Gels Containing Urea-- UDG (2.5 µg) was mixed with Ugi (1 µg) in a 15-µl volume consisting of 20 mM Tris-HCl, pH 7.5, and incubated for 15 min at room temperature and then for 15 min on ice (29, 30). Subsequently, loading dye (5 µl) consisting of 50 mM Tris-HCl, pH 6.8, and 10% (v/v) glycerol, and 0.1% bromphenol blue was added to the reaction, and it was subjected to electrophoresis on 10-cm-long 15% polyacrylamide (19:1 cross-linking) gels of 0.75-mm thickness without or with 2, 4, 6, or 8 M urea. The electrophoresis at ~10 V/cm was carried out using running buffer (25 mM Tris-base, 192 mM glycine, pH 8.8). The proteins were visualized by Coomassie Blue staining of the gels.

Bicistronic Constructs for Overproduction of UDG-Ugi Complexes-- Bicistronic constructs of various UDG mutants and Ugi were generated in pTrc99c exactly as described (30) except that the open reading frames of the Leu191 mutants (pTrc99c constructs) were amplified by polymerase chain reaction using Pfu DNA polymerase and the gene-specific forward (5'-CGGAATTCCATGGCTAACGAATTAACC-3') and reverse (5'-GGAATTCCTATTACTCACTCTCTGCC-3') primers. Template DNA (50 ng) and 20 pmol of the forward and the reverse primers were utilized in polymerase chain reaction under the following cycling conditions. The initial denaturation temperature was 95 °C for 1 min followed by 25 cycles of repeated denaturation at 95 °C for 45 s, annealing for 30 s at 50 °C, and extension at 72 °C for 1 min. The final extension was allowed at 72 °C for 10 min. To generate the bicistronic constructs in T7 RNA polymerase-based expression vector, NcoI-HindIII fragments harboring the complete bicistron from the pTrc99c constructs were subcloned into similarly digested pET11d (26).

Direct Analysis of the in Vivo Formed UDG-Ugi Complexes-- Overexpression of UDG and Ugi was achieved from the pTrc99c-based expression constructs exactly as described (30). The cell-free extracts were analyzed on 15% polyacrylamide (19:1 cross-linking) gels with or without 6 or 8 M urea.

Purification of the UDG-Ugi Complexes-- The purification of the complexes of Ugi with the wild type and mutant UDGs was achieved by the protocol described earlier (30) using the pET11d-based constructs. Due to the high level expression from the T7 RNA polymerase based vector, chromatography on DEAE-Sephacel and Mono-Q columns was found to be unnecessary. The purified complexes were quantified (27) and stored in 20 mM Tris-HCl, pH 7.4, at -20 °C.

Isothermal Denaturation of the UDG-Ugi Complexes in Urea-- UDG, Ugi, or the in vivo formed complexes of UDG and Ugi (1-2.5 µM) were incubated at 25 ± 2 °C for 4 h in the absence or presence of 2, 4, 6, and 8 M urea in 1 ml of 20 mM Tris-HCl, pH 7.4. Intrinsic fluorescence (tryptophan) changes were measured using a Jasco FP777 spectrofluorimeter with a thermostat cell holder using a cuvette of 1-cm path length and slit widths of 5 nm for excitation and emission. Appropriate buffer controls were also scanned and subtracted to arrive at the fluorescence intensity values. The excitation wavelength was 280 nm, and the corresponding emission spectra were recorded between 300 and 400 nm.

For detailed analysis of Ugi complexes with the wild type and the L191G UDGs, fluorescence intensities were measured at 332.5 and 326.5 nm, respectively. These wavelengths showed maximal fluorescence difference between the native (untreated sample) and the 8 M urea-treated samples. The emission intensities were converted into the relative fluorescence changes and plotted against the corresponding urea concentration. The relative fluorescence changes were calculated as (Ff - Fu)/Ff, where Ff corresponds to the fluorescence intensity of the native proteins and Fu corresponds to the fluorescence intensity of the urea-treated proteins (31).

Thermal Stability-- Thermal denaturation of the various Ugi complexes with UDGs (wild type, L191G, L191A, L191V, and L191F) was performed in a spectrophotometer (DU600; Beckman) as described (32). The concentration of the protein used was 1 µM and the absorbance changes were monitored at 287 nm. The first derivative of the thermal denaturation profile obtained using the software supplied with the instrument was used to evaluate the apparent transition temperatures for the proteins. The apparent denaturation temperature (apparent Tm) is defined as the temperature at which half of the protein is in the denatured state.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hyperexpression and Purification of the UDG Mutants-- Our repeated attempts to overproduce the UDG mutants at Leu191 using the pTrc99c-based expression constructs in E. coli BW310 (ung-) remained unsuccessful. Therefore, we used the T7 RNA polymerase-based expression constructs in E. coli BL21 (DE3). The pET11d-based constructs afforded hyperexpression of UDG even in the absence of induction. SDS-polyacrylamide gel analysis (33) revealed that the high level expression and the use of various column chromatography steps (see "Materials and Methods") yielded UDGs that were purified to apparent homogeneity (Fig. 1A). Although the E. coli BL21 (DE3) is wild type for ung, it has been shown that the UDG preparations obtained from such overexpression constructs are essentially free from the host chromosomal background (34).2 The T7 polymerase based expression systems for E. coli UDG mutants have been used in other studies as well (16, 35). Time course experiments (Fig. 1B) wherein a single-stranded DNA oligomer (SSU9) was used as substrate showed that the wild type and the L191V and L191F mutants were comparable in their uracil excision activity. However, compared with the wild type UDG, the L191A and L191G mutants retained about 10 and 1% enzymatic activity, respectively.


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Fig. 1.   A, analysis of purified UDGs (2.0 µg each) on a 15% polyacrylamide gel containing 0.1% SDS. Lane 1, WT; lane 2, L191G; lane 3, L191A; lane 4, L191V; lane 5, L191F. B, excision of uracil from single-stranded uracil-containing oligomer (SSU9) by the wild type and the mutant UDGs. The reactions were carried out as described under "Materials and Methods" for the indicated times with wild type (), or the mutant UDGs, L191G (open circle ), L191A (black-down-triangle ), L191V (down-triangle), and L191F (black-square).

Analysis of UDG-Ugi Complexes-- Ugi, encoded by the B. subtilis phage, PBS-1 or -2, is a proteinaceous inhibitor of UDG and is an exceptional mimic of the DNA substrate. Co-crystals of UDG-Ugi show that the side chain Leu191 of UDG fits into a hydrophobic cavity of Ugi (15, 17). It was therefore of interest to us to analyze the stability of the complexes of Ugi with the UDG mutants with varying length of the side chains at position 191. Since the B. subtilis UDG, which is a natural target of Ugi, contains phenylalanine in place of the Leu191 in the E. coli UDG, we were also interested in the study of L191F for its ability to interact with Ugi.

Fig. 2A shows electrophoretic analysis of the in vitro formed complexes of Ugi with UDGs on a native polyacrylamide gel. UDG (pI, 6.6; molecular mass, 25.6 kDa) migrates slowest (lane 1), and Ugi (pI, 4.2; molecular mass, 9.4 kDa) migrates fastest (lane 6). The complex of the two proteins (pI, 4.9; molecular mass, 35 kDa) migrates with intermediate mobility (Fig. 2A, lanes 2-6). The electrophoretic analysis of the native gel did not reveal any differences between the complexes of Ugi with the wild type or the mutant UDGs. The wild type UDG forms a physiologically irreversible complex with Ugi (29). In fact, the interaction between UDG and Ugi is so strong that, unless heated, the complex does not dissociate even after several hours of incubation in M urea. Therefore, to study the effect of mutations at Leu191, we developed a urea-polyacrylamide gel system (Fig. 2, B-E) to discriminate the UDG-Ugi complexes based on their relative stability. In the presence of 2 or 4 M urea, UDG (lane 1) and Ugi (lane 7) begin to migrate as diffuse bands (Fig. 2, B and C), suggesting urea-mediated unfolding of these proteins. However, upon further increase in urea concentration (6 and 8 M), their mobility on the gel was relatively more compact (Fig. 2, D and E). This is most likely, because with the increase in urea concentration, the unfolding of the proteins was complete. More importantly, the complex of wild type UDG with Ugi migrated as a sharp band even on a gel containing 8 M urea (compare lanes 2 in Fig. 2, A-E). This observation suggests that while the UDG and Ugi are individually susceptible to urea-mediated unfolding, the complex of the two becomes impervious to urea under these conditions. However, under the same conditions, the complex of Ugi with L191G UDG was more susceptible to urea-induced perturbation, and it began to dissociate in 4 M urea gel (appearance of smear in Fig. 2C, lane 3), and the dissociation was complete in 6 M urea gel (Fig. 2D, lane 3). Unlike the L191G mutant, the complexes of L191A and L191V with Ugi remain intact at 4 M urea (Fig. 2C, lanes 4 and 5) but begin to dissociate in 6 M urea (Fig. 2D, lanes 4 and 5). On the 8 M urea gel, while the complex of Ugi with the wild type and the L191F UDGs showed a detectable smear, the other complexes dissociated completely. Furthermore, in the 8 M urea gel (Fig. 2E), we observed that from among the complexes of Ugi with the L191G, L191A, and L191V mutants, the Ugi band that resulted upon the dissociation of the respective complexes (Fig. 2E) migrated fastest in lane 3 (L191G), intermediately in lane 4 (L191A), and slowest in lane 5 (L191V). On the contrary, the mobility of the smear corresponding to UDG was maximum for the L191V, intermediate for L191A, and least for L191G. Interestingly, the Ugi band in lane 3 (L191G) migrated just slightly slower than the Ugi control (lane 7), suggesting that the L191G-Ugi dissociated soon after its entry into the gel. Taken together, the use of these urea-polyacrylamide gels enabled us to monitor the progressive real time dissociation of the UDG-Ugi complexes, and the relative stability of the Ugi complexes with the various UDGs could be established in the following descending order, wild type = L191F > L191V > L191A > L191G. This order of stability of the complexes demonstrates that replacement of Leu191 with a shorter side chain (L191A) or its abolition (L191G) resulted in a weaker interaction with Ugi.


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Fig. 2.   Analysis of the UDG-Ugi complexes on 15% polyacrylamide gels in the absence (A) or presence of 2 M (B), 4 M (C), 6 M (D), and 8 M (E) urea. Lane 1, E. coli UDG; lanes 2-6, complexes of Ugi with the wild type, L191G, L191A, L191V, and L191F UDGs, respectively; lane 7, Ugi. The various arrows used to denote UDG, Ugi, or the complex of the two are shown in the last panel.

Analysis of the in Vivo Formed UDG-Ugi Complexes on Urea Gels-- We described a bicistronic construct for formation of the UDG-Ugi complexes within the cellular milieu (30). Using similar bicistronic constructs, we have analyzed the stability of the in vivo formed complexes of Ugi with the Leu191 mutants without subjecting them to any purification. As reported earlier (30) and also seen from the presence of free Ugi (along with the UDG-Ugi complexes) on the native gel (Fig. 3), the bicistronic constructs overproduce Ugi in excess of 1:1 molar stoichiometry with UDG. The analysis of the cell-free extracts on the urea-polyacrylamide gels shows that, similar to the in vitro formed complexes, the L191G complex dissociated in 6 M urea, whereas the complexes of L191A and L191V dissociated in 8 M urea. Also, the relative mobility of the dissociated Ugi (Fig. 3, 8 M urea) reaffirmed the relative stability of the complexes as determined for the in vitro formed complexes (Fig. 2). Since, in this experiment, total cell-free extracts were used, relative mobility of different UDG bands after dissociation from the respective complexes could not be ascertained. More importantly, this analysis demonstrates that the urea-polyacrylamide gels afforded direct analysis of the in vivo formed UDG-Ugi complexes from the cell-free extracts.


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Fig. 3.   Analysis of the in vivo formed complexes of Ugi with UDG. The cell-free extracts from the transformants harboring the bicistronic construct for Ugi and the various mutants of UDG were analyzed on 15% polyacrylamide gels containing no urea or 6 or 8 M urea. Lane 1, UDG (wild type)-Ugi complex; lanes 2-5, ~10 µg of total proteins from the transformants harboring the bicistronic construct for L191G, L191A, L191V, and L191F, respectively; lane 6, Ugi. The various arrows used to denote Ugi and its complex with UDG are the same as in Fig. 2.

Spectrofluorometric Analysis of Urea-induced Unfolding of the UDG-Ugi Complexes-- In these experiments, we have utilized the changes in intrinsic fluorescence (tryptophan) as a correlate to analyze the structural changes in the proteins upon isothermal denaturation with varying concentrations of urea. As seen in Fig. 4A, incubation of Ugi and UDG with different concentrations of urea (2-8 M) resulted in the successive enhancement (for Ugi) and decrease (for UDG) of the fluorescence intensities. Concomitant with the fluorescence change, there was a red shift in the fluorescence spectra. These changes in the intrinsic fluorescence and the red shift of the spectra are indicative of urea-induced unfolding of proteins. Interestingly, when the complex of Ugi with wild type UDG was subjected to a similar analysis, no changes in the intrinsic fluorescence were observed (Fig. 4B, panel W. T.), suggesting that while the individual proteins were highly susceptible to urea-mediated unfolding, the complex of the two was recalcitrant to such a treatment. However, when the other complexes were subjected to similar analysis, at M urea, the complex with L191G showed fluorescence quenching along with a red shift in the spectrum. This spectrum was not affected further at 8 M urea. Similar changes in the spectral profile for the complexes with L191A and L191V were observed upon incubation in 8 M urea. Strikingly, these observations are in complete accordance with the analysis of these complexes on the urea-polyacrylamide gels (Fig. 2, A-E) and suggest that the intrinsic fluorescence quenching and the concomitant red shift in the spectra correlate with the dissociation of the UDG-Ugi complex. However, the complex of Ugi with L191F mutant showed a gradual decrease of the intrinsic fluorescence upon incubation with urea, but this was not associated with a red shift of the fluorescence spectra. Considering that in our urea-polyacrylamide gel, this complex was maintained even at 8 M urea, the fluorescence differences may indicate minor perturbations in this complex that do not result in its dissociation (see "Discussion").


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Fig. 4.   A, intrinsic fluorescence (Trp) changes in UDG, and Ugi (1 µM) in response to urea. The excitation wavelength was 280 nm, and the emission spectra were recorded between 300 and 400 nm. B, intrinsic fluorescence (Trp) changes in UDG-Ugi complexes (2.5 µM) in response to urea. Excitation wavelength was 280 nm, and the emission spectra were recorded between 300 and 400 nm. A-E, complexes of Ugi with WT, L191G, L191A, L191V, and L191F UDGs, respectively. The various concentrations of urea with which they were incubated are as shown in the lower right. C, difference fluorescence spectra showing changes in the intrinsic fluorescence (tryptophan) of the UDG-Ugi complexes. The complexes were either not treated with urea or treated with the indicated concentration of urea in 20 mM Tris-HCl (pH 7.4) at 25 ± 2 °C for 4 h before recording the changes in fluorescence emission intensities at 332.5 and 326.5 nm, respectively for complexes of Ugi with the wild type and the L191G UDG. These wavelengths corresponded to lambda em at which maximum difference in fluorescence intensities were observed between the native (0 M urea) and treated (8 M urea) proteins. The emission intensities were converted to relative fluorescence changes and plotted against the corresponding denaturant concentrations. The filled and the open circles show relative fluorescence changes of the Ugi complexes with the wild type and the L191G UDGs, respectively.

Detailed analysis of urea-induced equilibrium unfolding studies for the Ugi complexes with wild type and the L191G UDGs was also carried out by monitoring fluorescence changes at a single wavelength at which the difference in the fluorescence intensity is maximum between 0 and 8 M urea concentrations. These wavelengths for the Ugi complexes with the wild type and the L191G mutant were found to be 332.5 and 326.5 nm, respectively. As seen in Fig. 4C, consistent with the observations made in earlier experiments, while the wild type complex did not register a significant change and responded as a fully folded entity at all concentrations of urea, the L191G (UDG)-Ugi complex showed a smooth transition between the folded and the unfolded form at around 4.5 M urea. This analysis further supports the observations made in Figs. 2 and 4B.

Determination of Melting Temperatures of Various UDG-Ugi Complexes-- To further analyze the effect of mutations at Leu191, we determined the apparent Tm values of the various UDG-Ugi complexes (Table I). The apparent Tm values of the Ugi complexes with the wild type and the L191F UDGs were ~80 °C. However, the apparent Tm values of the complexes formed by the L191G, L191A, and L191V mutants of UDG were lower (~71-74 °C). Minor differences in the Tm values of the complexes formed by L191G, L191A, and L191V UDGs do not allow us to discriminate among them for their relative thermal stability. Nevertheless, the Tm analysis does highlight the role of the two naturally occurring amino acids, leucine (in most of the UDGs) and phenylalanine (in the UDG from B. subtilis) at position 191 (or its equivalent) in forming a highly stable UDG-Ugi complex.

                              
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Table I
Apparent Tm (°C) values of wild type UDG-Ugi, L191G-Ugi, L191A-Ugi, L191V-Ugi, and L191F-Ugi complexes

Kinetics of Uracil Excision from SSU9 and AU9-- Three-dimensional structure determinations of the co-crystals of UDG with Ugi suggested that Ugi is a transition state substrate mimic (17). Our analysis of the Ugi complexes with the UDG mutants such as the L191G and L191A showed that obliteration or reduction in the length of the Leu191 side chain resulted in a decrease in the stability of the complex. Also, as seen in Fig. 1B, these are also the mutants that showed compromised UDG activity. Further, based on an earlier study which proposed the "push-pull" mechanism for localization of the uridine into the active site pocket of the human UDG (36), it was of interest to analyze the effect of these two mutations at Leu191 on the kinetic parameters of uracil excision. The two substrates harboring dU in the same sequence context but in different structural contexts were used for this analysis. The SSU9 substrate harbors the dU residue in the single-stranded ("unstructured") context, and the AU9 hairpin harbors the dU residue in the double-stranded context in the middle of the stem region. In both substrates, dU is located at the 9th position from the 5'-end.

As predicted from the push-pull model for target site recognition, the kinetic parameters (Table II) of uracil excision from SSU9 and AU9 substrates using L191G and L191A mutants showed that they were compromised in their uracil excision activity. The ratio of relative efficiency (Vmax/Km) by which the uracil was excised from the single- and double-stranded DNA by the wild type UDG was ~10.4. More importantly, the ratio of relative efficiency of uracil excision from SSU9 and AU9 was maintained even for the L191G and the L191A mutants (~8.7 and 9.5, respectively). Thus, while the L191G and L191A were compromised in the uracil excision, the relative utilization of single- and double-stranded substrates by them was similar to that of the wild type. These analyses suggest that changes at Leu191 must be affecting a common step in the catalysis of uracil excision from the single- and double-stranded substrates.

                              
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Table II
Kinetic parameters for uracil excision from SSU9 and AU9


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The ability of uracil DNA glycosylases to use both the single- and double-stranded DNA as substrates makes these enzymes an ideal system to study the mechanism of damage removal from DNA. Furthermore, since the conserved UDGs from different sources form a highly stable complex with the B. subtilis phage (PBS-1 or -2)-encoded proteinaceous inhibitor (Ugi), these proteins have also been of interest to understand the mechanism of protein-protein interaction. Due to their small sizes, these proteins have been amenable for three-dimensional structural determinations by x-ray crystallography and NMR. Several crystal structures have shown Ugi to be a mimic of the DNA substrate. Remarkably, the recent comparative analysis of the UDG-Ugi structure with the UDG-DNA structure demonstrates that Ugi mimics the transition state of the DNA bound to UDG (17). One of the contacts that UDG establishes with Ugi is through its highly conserved Leu191 into the hydrophobic pocket of Ugi. This pocket is shaped by the side chains of Met24, Val29, Val32, Ile33, Val43, Met56, Leu58, and Val71 between the beta 1 edge of the antiparallel beta -sheet and the alpha 2 helix. This cavity in Ugi is functionally similar to the void generated in the DNA base stack upon extrahelical localization of uridine.

We have investigated the effect of mutating Leu191 of E. coli UDG on its ability to form a complex with Ugi and on the efficiencies of uracil excision from the single- and double-stranded DNA substrates. The mutational analysis shows that shortening or deletion of the Leu191 side chain results in UDGs whose complexes with Ugi are less stable than the one formed by the wild type UDG. Both the electrophoretic analysis on urea gels (Figs. 2 and 3) and apparent Tm determinations (Table I) show that the L191F mutant formed a complex with Ugi, which was as stable as that of the wild type UDG. These observations are consistent with the prediction that bigger side chains can be accommodated in the Ugi pocket (17). Thus, the reason(s) for slight decrease in the intrinsic fluorescence of the L191F mutant complex with Ugi in the presence of urea (Fig. 4B) are unclear at present. However, it may be noted that, in E. coli UDG, the Leu191 is differently oriented than that from human UDG, and the context of the sequence within the DNA intercalation loop is likely to be important for its docking into the hydrophobic cavity of Ugi (15, 17). It would be interesting to study the urea-induced intrinsic fluorescence changes in the complex of B. subtilis UDG, which is the biological ligate of Ugi and where phenylalanine occurs naturally at this position. The significance of the differently oriented HPSPLS loop is also evident from the observation that leucine to alanine mutation in the HPSPLS motif in the context of E. coli UDG still retained about 10% enzymatic activity (Table II), whereas the same mutation in the context of human UDG resulted in severe loss of the activity (1% as active as the wild type enzyme) (37).

Cocrystal structure of an engineered human UDG (L272R/D145N) with the in situ cleaved products led the authors to suggest the push and pull mechanism for localization of the uracil into the active site pocket of the enzyme. It was proposed that the Leu272 side chain (L of the HPSPLS motif) inserted into the base stack through the minor groove, causing extrahelical localization ("push") of the target uridine, which was then accommodated ("pull") into the active site pocket by establishing uracil-specific contacts (36). However, subsequently from a high resolution crystal structure (37), it was proposed that the uracil localization into the active site pocket of the enzyme utilized a concerted mechanism of "pinch, push, and pull." The serine residues of the highly conserved HPSPLS and PPPS motifs hydrogen-bonded with the phosphates flanking the target uridine, resulting in compression and kinking of the DNA backbone in the vicinity of the lesion, leading to the expulsion of the target base. More interestingly, it was seen that even in the L272A mutant containing a shorter side chain, which was unable to reach into the base stack, the nucleotide flipping occurred, suggesting that Leu272 was not absolutely critical for the localization of the target uridine into the active site pocket (37). Our results on the kinetics of uracil excision from both the single- and double-stranded substrates by the E. coli UDG Leu191 mutants are in complete agreement with the pinch, push, and pull mechanism of the damage recognition. Thus, shortening of the Leu191 side chain (L191A) or deleting it altogether (L191G) decreased the uracil excision efficiency to ~10 and 1%, respectively (Table II, Fig. 1B).

However, from a different perspective, the double-stranded DNA involves a defined network of base-stacking and hydrogen-bonding interactions; in energetic terms, the extrahelical localization of uracil from double-stranded substrates would be more demanding compared with the single-stranded substrates. Therefore, it may be argued that the penalty for shortening the Leu191 side chain (L191A) or deleting it altogether (L191G) will be higher for uracil excision from the double-stranded substrates as compared with the single-stranded substrate. Interestingly, we observed that although the L191A and L191G showed decreased efficiency of uracil excision, the relative ratio of uracil excision from the single-stranded (SSU9) and double-stranded (AU9) substrates was unaffected (compared with wild type, the relative ratios of uracil excision from single-stranded versus double-stranded substrate for L191A and L191G were 0.91 and 0.84, respectively). Thus, our results point out to the role of Leu191 at a step utilized by both the single- and double-stranded substrates in common. These observations can be easily rationalized if the interaction between the Ser88, Ser189, and Ser192 and the phosphates flanking the target uridine (37-39), which lead to the compression (the "pinch" step) of the sugar phosphate backbone, was the first event to take place in the substrate recognition. The kinking of the backbone would result in destabilization of the hydrogen bonds of the target uracil with its complement A or G. Thus, much of the discrimination that is seen in utilization of substrates containing uracil in different structural contexts (23, 40) should occur at the step of interaction of UDG with the DNA backbone. And during catalysis, if the Leu191 participated subsequent to this step, it will not discriminate between the single- and double- stranded substrates.

Since the "push" step is not absolutely required for the localization of the target uridine into the active site pocket of the enzyme, based on the observation that the L191G (which lacked the side chain) was highly compromised in its enzymatic activity, we envisage an additional role of leucine of the HPSPLS motif. Once the uridine is localized into the active site pocket, this leucine could be important in the stabilization of the productive enzyme-substrate complex by inserting its side chain into the void generated between the flanking bases subsequent to extrahelical localization of the target uridine. Thus, a parallel could be drawn for the role of Leu191 in imparting stability to the UDG-Ugi complex by its docking into the hydrophobic cavity of Ugi on one hand and stabilization of the productive enzyme-substrate complex by inserting its side chain between the flanking bases on the other (Fig. 5, a and b). This stabilization would be impaired for the mutant L191A (c and d) and abolished altogether in the case of L191G (e and f). In fact, the surface plasmon resonance study with the wild type and the L272A of human UDG (equivalent of L191A of E. coli UDG) did show that while the "on rates" of enzyme binding to a nonhydrolyzable uracil (4'-S-dU) in a double-stranded (in a U:A base pair) substrate for the wild type and the L272A mutant were similar (ka, 0.16 versus 0.15 ×106/M/s), L272A showed faster "off rates" as compared with the wild type UDG (0.13 versus 0.05/s) (37). The Km measurements for the L191G mutant (Table II) are also consistent with these studies.


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Fig. 5.   Stereo view models highlighting the interaction of the residue 191 (within the DNA intercalation loop) of E. coli UDG (in red) with Ugi (a, c, and e) and the uncleaved DNA substrate (b, d, and f) generated by the INSIGHTII package using the coordinates from crystal structures of E. coli UDG-Ugi (Protein Data Bank code 1UUG; Ref. 17) and human UDG-DNA (Protein Data Bank code 1EMH; Ref. 41). The side chain of Leu191 is shown inserting into the hydrophobic pocket of Ugi (a) and into the void between the neighboring bases generated upon flipping out of the target uridine (b). c and d, same as a and b except that the leucine side chain has been replaced with alanine, which results in its partial entry into the respective hydrophobic cavities. e and f, same as a and b except that the leucine side chain has been changed to glycine, which results in the complete abolition of the hydrophobic contact seen before between the Leu191 and Ugi or the DNA. The Ugi and the target DNA strand are shown in green. The DNA complementary to the target strand is shown in yellow.

Finally, in this study, we have developed a polyacrylamide gel system that has allowed us to discriminate among the UDG-Ugi complexes on the basis of their stability in urea. The results obtained from the urea-polyacrylamide gels were in complete agreement with those obtained from the biophysical methods (Fig. 4). More importantly, the urea gel analysis not only used much smaller amounts of the protein but also permitted the analysis of the UDG-Ugi complexes directly from the cell-free extracts. Also, the observation that the relative stability of the complexes of Ugi with WT, L191G, L191A, L191V, and L191F UDGs could be ascertained from a single gel (Figs. 2 and 3) highlights the potential of this gel system in facile screening of a large number of mutants of the two interacting proteins.

    ACKNOWLEDGEMENT

We thank K. Saikrishnan for invaluable help in generating the models shown in Fig. 5.

    FOOTNOTES

* This work was supported in part by research grants from the Council of Scientific and Industrial Research (CSIR), the Department of Biotechnology, and the Department of Science and Technology, New Delhi, India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a senior research fellowship of CSIR.

§ To whom correspondence should be addressed. Tel.: 91-80-309-2686; Fax: 91-80-360-2697 or 91-80-360-0683; E-mail: varshney@ mcbl.iisc.ernet.in.

Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M011166200

2 P. Handa, S. Roy, and U. Varshney, unpublished data.

    ABBREVIATIONS

The abbreviations used are: UDG, uracil DNA glycosylase; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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