The Role of Leucine 191 of Escherichia coliUracil DNA Glycosylase in the Formation of a Highly Stable Complex with the Substrate Mimic, Ugi, and in Uracil Excision from the Synthetic Substrates
2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês
10.1074/jbc.m011166200
ISSN1083-351X
AutoresPriya Handa, Sudipta Roy, Umesh Varshney,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoUracil 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 the187HPSPLS192 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 Leu191with 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. 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 the187HPSPLS192 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 Leu191with 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. uracil DNA glycosylase wild type 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 (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4224) Google Scholar). Uracil is a natural component of RNA but can occur in the DNA by spontaneous deamination of an inherently unstable base, cytosine (2Friedberg E.C. Walker G.C. Wolfram S. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington D. C.1995: 1-58Google Scholar). Occasional incorporation of dUMP, in place of dTMP, during DNA synthesis is yet another way by which uracil can arise in DNA (3Tye B.K. Nyman P.O. Lehaman I.R. Hochhauser S. Weiss B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 154-157Crossref PubMed Scopus (224) Google Scholar). If left unrepaired, in a U:G mismatch, uracil is promutagenic and can lead to GC→AT 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 (4Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-369Crossref PubMed Scopus (97) Google Scholar). 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 (5Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3649-3653Crossref PubMed Scopus (462) Google Scholar, 6Lindahl T. Ljungquist S. Siegert W. Nyberg B. Sperens B. J. Biol. Chem. 1977; 252: 3286-3294Abstract Full Text PDF PubMed Google Scholar). 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 (7Aravind L. Koonin E.V. Genome Biol.2000http://www.genomebiology.com/2000/1/4/research/0007/Google Scholar, 8Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-15Crossref PubMed Scopus (721) Google Scholar). 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 (9Wang Z. Mosbaugh D.W. J. Biol. Chem. 1989; 264: 1163-1171Abstract Full Text PDF PubMed Google Scholar, 10Nagelhus T.A. Haug T. Singh K.K. Keshav K.F. Skorpen F. Otterlei M. Bharati S. Lindmo T. Benichou S. Benarous R. Krokan H.E. J. Biol. Chem. 1997; 272: 6561-6566Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11Purnapatre K. Handa P. Venkatesh J. Varshney U. Nucleic Acids Res. 1999; 27: 3487-3492Crossref PubMed Scopus (22) Google Scholar, 12Otterlei M. Warbrick E. Nagelhus T.A. Haug T. Slupphaug G. Akbari M. Aas P.A. Steinsbekk K. Bakke O. Krokan H.E. EMBO J. 1999; 18: 3834-3844Crossref PubMed Scopus (294) Google Scholar). 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 (13Mol C.D. Arvai A.S. Slupphaug G. Kavli B. Krokan H.E. Tainer J.A. Cell. 1995; 80: 869-878Abstract Full Text PDF PubMed Scopus (338) Google Scholar, 14Savva R. McAuley-Hecht K. Brown T. Pearl L. Nature. 1995; 373: 483-493Crossref Scopus (385) Google Scholar). The crystal structures of Escherichia coli UDG (15Ravishankar R. Bidya Sagar M. Roy S. Purnapatre K. Handa P. Varshney U. Vijayan M. Nucleic Acids Res. 1998; 26: 4880-4887Crossref PubMed Scopus (54) Google Scholar, 16Xiao G. Tordova M. Jagadeesh J. Drohat A.C. Stivers J.T. Gilliland G.L. Proteins Struct. Funct. Genet. 1999; 35: 13-24Crossref PubMed Scopus (106) Google Scholar, 17Putnam C.D. Shroyer M.J.N. Lundquist A.J. Mol C.D. Arvai A.S. Mosbaugh D.W. J. Mol. Biol. 1999; 287: 331-346Crossref PubMed Scopus (113) Google Scholar) 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) inE. coli UDG show subtle conformational differences when compared with those in human UDG (15Ravishankar R. Bidya Sagar M. Roy S. Purnapatre K. Handa P. Varshney U. Vijayan M. Nucleic Acids Res. 1998; 26: 4880-4887Crossref PubMed Scopus (54) Google Scholar, 16Xiao G. Tordova M. Jagadeesh J. Drohat A.C. Stivers J.T. Gilliland G.L. Proteins Struct. Funct. Genet. 1999; 35: 13-24Crossref PubMed Scopus (106) Google Scholar). The cocrystal structures of the human herpes simplex virus-1 and E. coli UDGs with Ugi (15Ravishankar R. Bidya Sagar M. Roy S. Purnapatre K. Handa P. Varshney U. Vijayan M. Nucleic Acids Res. 1998; 26: 4880-4887Crossref PubMed Scopus (54) Google Scholar, 17Putnam C.D. Shroyer M.J.N. Lundquist A.J. Mol C.D. Arvai A.S. Mosbaugh D.W. J. Mol. Biol. 1999; 287: 331-346Crossref PubMed Scopus (113) Google Scholar, 18Savva R. Pearl L.H. Nat. Struct. Biol. 1995; 2: 752-757Crossref PubMed Scopus (86) Google Scholar, 19Mol C.D. Arvai A.S. Sanderson R.J. Slupphaug G. Kavli B. Krokan H.E. Mosbaugh D.W. Tainer J.A. Cell. 1995; 82: 701-708Abstract Full Text PDF PubMed Scopus (232) Google Scholar) 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. coliUDG and eight hydrophobic residues of Ugi (Met24, Val29, Val32, Ile33, Val43, Met56, Leu58, and Val71) in a cavity burrowed between the α2and the antiparallel β sheet of Ugi and (ii) the electrostatic interactions between the acidic residues of the β1 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 (17Putnam C.D. Shroyer M.J.N. Lundquist A.J. Mol C.D. Arvai A.S. Mosbaugh D.W. J. Mol. Biol. 1999; 287: 331-346Crossref PubMed Scopus (113) Google Scholar). 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 (20Lundquist A.J. Beger R.D. Bennett S.E. Bolton P.H. Mosbaugh D.W. J. Biol. Chem. 1997; 272: 21408-21419Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). 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 vivoconditions. 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. 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. Oligonucleotides (10 pmol) were 5′-32P-end labeled using 10 μCi of [γ-32P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase in 10-μl reaction volumes (21Chaconas G. van de Sande J.H. Methods Enzymol. 1980; 65: 75-85Crossref PubMed Scopus (191) Google Scholar) and purified by chromatography on Sephadex G-50 minicolumns (22Kumar N.V. Varshney U. Nucleic Acids Res. 1994; 22: 3737-3741Crossref PubMed Scopus (24) Google Scholar). This procedure routinely resulted in labeling efficiency of ∼106 cpm/pmol. ForKm and Vmax determinations, radiolabeled oligonucleotides were mixed with cold substrates such that the contribution from labeled substrates was much less than 1% (23Kumar N.V. Varshney U. Nucleic Acids Res. 1997; 25: 2336-2343Crossref PubMed Scopus (30) Google Scholar). A DNA fragment (∼800 base pairs) harboring the sequence downstream of the BamHI site within the ung gene was excised from pTZUng4 usingBamHI (24Varshney U. Hutcheon T. van de Sande J.H. J. Biol. Chem. 1988; 263: 7776-7784Abstract Full Text PDF PubMed Google Scholar) and subcloned into a similarly digested pTZ19R to generate pTZUng2B′. This recombinant plasmid is similar to pTZUng2B (24Varshney U. Hutcheon T. van de Sande J.H. J. Biol. Chem. 1988; 263: 7776-7784Abstract Full Text PDF PubMed Google Scholar) 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 (25Handa P. Varshney U. Ind. J. Biochem. Biophys. 1998; 35: 63-66PubMed Google Scholar). 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 usingPfu 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 aHindIII 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 (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The expression constructs in pTrc99c and pET11d thus obtained were reaffirmed by nucleotide sequencing. 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 (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Cells were harvested, and UDG was purified by a modification of the procedure described earlier (6Lindahl T. Ljungquist S. Siegert W. Nyberg B. Sperens B. J. Biol. Chem. 1977; 252: 3286-3294Abstract Full Text PDF PubMed Google Scholar). 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 mmNaCl, 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 mmHepes, 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 0–1m 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 (27Sedmak J.J. Grossberg S.E. Anal. Biochem. 1977; 79: 544-552Crossref PubMed Scopus (2471) Google Scholar), and stored at −20 °C. 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). 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-8m urea gels. The bands corresponding to the product and the remaining substrate were quantified by using a BioImage Analyser (Fuji). Values of Km and Vmaxwere determined from Hofstee plots (28Dowd J.E. Riggs D.J. J. Biol. Chem. 1965; 240: 863-869Abstract Full Text PDF PubMed Google Scholar) of at least two independent experiments. UDG (2.5 μg) was mixed with Ugi (1 μg) in a 15-μl volume consisting of 20 mmTris-HCl, pH 7.5, and incubated for 15 min at room temperature and then for 15 min on ice (29Bennett S.E. Mosbaugh D.W. J. Biol. Chem. 1992; 268: 22512-22521Abstract Full Text PDF Google Scholar, 30Roy S. Purnapatre K. Handa P. Boyanapalli M. Varshney U. Protein Expression Purif. 1998; 13: 155-162Crossref PubMed Scopus (17) Google Scholar). 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 of various UDG mutants and Ugi were generated in pTrc99c exactly as described (30Roy S. Purnapatre K. Handa P. Boyanapalli M. Varshney U. Protein Expression Purif. 1998; 13: 155-162Crossref PubMed Scopus (17) Google Scholar) except that the open reading frames of the Leu191 mutants (pTrc99c constructs) were amplified by polymerase chain reaction usingPfu 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 (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Overexpression of UDG and Ugi was achieved from the pTrc99c-based expression constructs exactly as described (30Roy S. Purnapatre K. Handa P. Boyanapalli M. Varshney U. Protein Expression Purif. 1998; 13: 155-162Crossref PubMed Scopus (17) Google Scholar). The cell-free extracts were analyzed on 15% polyacrylamide (19:1 cross-linking) gels with or without 6 or 8 m urea. The purification of the complexes of Ugi with the wild type and mutant UDGs was achieved by the protocol described earlier (30Roy S. Purnapatre K. Handa P. Boyanapalli M. Varshney U. Protein Expression Purif. 1998; 13: 155-162Crossref PubMed Scopus (17) Google Scholar) 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 (27Sedmak J.J. Grossberg S.E. Anal. Biochem. 1977; 79: 544-552Crossref PubMed Scopus (2471) Google Scholar) and stored in 20 mm Tris-HCl, pH 7.4, at −20 °C. 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 murea 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 murea-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, whereFf corresponds to the fluorescence intensity of the native proteins and Fu corresponds to the fluorescence intensity of the urea-treated proteins (31Handa P. Acharya N. Thanedar S. Purnapatre K. Varshney U. Nucleic Acids Res. 2000; 28: 3823-3829Crossref PubMed Scopus (16) Google Scholar). 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 (32Rao J.V.K. Prakash V. Rao N., A. Savithri H.S. Eur. J. Biochem. 2000; 267: 5967-5976Crossref PubMed Scopus (28) Google Scholar). 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. Our repeated attempts to overproduce the UDG mutants at Leu191using the pTrc99c-based expression constructs in E. coliBW310 (ung−) remained unsuccessful. Therefore, we used the T7 RNA polymerase-based expression constructs in E. coliBL21 (DE3). The pET11d-based constructs afforded hyperexpression of UDG even in the absence of induction. SDS-polyacrylamide gel analysis (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar) 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.1 A). 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 (34Shroyer M. Bennett S.E. Putnam C.D. Tainer J.A. Mosbaugh D.W. Biochemistry. 1999; 38: 4834-4845Crossref PubMed Scopus (30) Google Scholar). 2P. Handa, S. Roy, and U. Varshney, unpublished data. The T7 polymerase based expression systems for E. coli UDG mutants have been used in other studies as well (16Xiao G. Tordova M. Jagadeesh J. Drohat A.C. Stivers J.T. Gilliland G.L. Proteins Struct. Funct. Genet. 1999; 35: 13-24Crossref PubMed Scopus (106) Google Scholar, 35Stivers J.T. Pankiewicz K.W. Watanabe K.A. Biochemistry. 1999; 38: 952-963Crossref PubMed Scopus (191) Google Scholar). Time course experiments (Fig.1 B) 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. 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 (15Ravishankar R. Bidya Sagar M. Roy S. Purnapatre K. Handa P. Varshney U. Vijayan M. Nucleic Acids Res. 1998; 26: 4880-4887Crossref PubMed Scopus (54) Google Scholar, 17Putnam C.D. Shroyer M.J.N. Lundquist A.J. Mol C.D. Arvai A.S. Mosbaugh D.W. J. Mol. Biol. 1999; 287: 331-346Crossref PubMed Scopus (113) Google Scholar). 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 theB. 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. 2 A 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. 2 A,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 (29Bennett S.E. Mosbaugh D.W. J. Biol. Chem. 1992; 268: 22512-22521Abstract Full Text PDF Google Scholar). 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 8m 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, Band C), suggesting urea-mediated unfolding of these proteins. However, upon further increase in urea concentration (6 and 8m), 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 4m urea gel (appearance of smear in Fig. 2 C,lane 3), and the dissociation was complete in 6m urea gel (Fig. 2 D, lane 3). Unlike the L191G mutant, the complexes of L191A and L191V with Ugi remain intact at 4 m urea (Fig.2 C, lanes 4 and 5) but begin to dissociate in 6 m urea (Fig. 2 D,lanes 4 and 5). On the 8 murea 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.2 E), 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. 2 E) migrated fastest in lane 3 (L191G), intermediately in lane 4 (L191A), and slowest inlane 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 inlane 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 relat
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