Substitutions at tyrosine 66 of Escherichia coli uracil DNA glycosylase lead to characterization of an efficient enzyme that is recalcitrant to product inhibition

Uracil DNA glycosylase (UDG), a ubiquitous and highly specific enzyme, commences the uracil excision repair pathway. Structural studies have shown that the tyrosine in a highly conserved GQDPY water-activating loop of UDGs blocks the entry of thymine or purines into the active site pocket. To further understand the role of this tyrosine (Y66 in Escherichia coli UDG), we have overproduced and characterized Y66F, Y66H, Y66L and Y66W mutants. The complexes of the wild-type, Y66F, Y66H and Y66L UDGs with uracil DNA glycosylase inhibitor (Ugi) (a proteinaceous substrate mimic) were stable to 8 M urea. However, some dissociation of the complex involving the Y66W UDG occurred at this concentration of urea. The catalytic efficiencies (V_max / K_m) of the Y66L and Y66F mutants were similar to those of the wild-type UDG. However, the Y66W and Y66H mutants were ~7- and ~173-fold compromised, respectively, in their activities. Interestingly, the Y66W mutation has resulted in an enzyme which is resistant to product inhibition. Preferential utilization of a substrate enabling a long range contact between the –5 phosphate (upstream to the scissile uracil) and the enzyme, and the results of modeling studies showing that the uracil-binding cavity of Y66W is wider than those of the wild type and other mutant UDGs, suggest a weaker interaction between uracil and the Y66W mutant. Furthermore, the fluorescence spectroscopy of UDGs and their complexes with Ugi, in the presence of uracil or its analog, 5-bromouracil, suggests compromised binding of uracil in the active site pocket of the Y66W mutant. Lack of inhibition of the Y66W UDG by apyrimidinic DNA (AP-DNA) is discussed to highlight a potential additional role of Y66 in shielding the toxic effects of AP-DNA, by lowering the rate of its release for subsequent recognition by an AP endonuclease.     

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

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.     

Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes.

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in eukaryotes and prokaryotes [1][2][3]. This enzyme removes uracil bases that are present in DNA as a result of either deamination of cytosine or misincorporation of dUMP instead of dTMP [4] [5], and it is the primary activity in the DNA base excision repair pathway. Although UDG activities have been shown to be present in several thermophiles [6][7][8], no sequences have been found that are complementary to the Escherichia coli ung gene, which encodes UDG [9]. Here, we describe a UDG from the thermophile Thermotoga maritima. The T. maritima UDG gene has a low level of homology to the E. coli G-T/U mismatch-specific DNA glycosylase gene (mug). The expressed protein is capable of removing uracil from DNA containing either a U-A or a U-G base pair and is heat-stable up to 75 degrees C. The enzyme is also active on single-stranded DNA containing uracil. Analogous genes appear to be present in several prokaryotic organisms, including thermophilic and mesophilic eubacteria as well as archaebacteria, the human-disease pathogens Treponema palladium and Rickettsia prowazekii, and the extremely radioresistant organism Deinococcus radiodurans. These findings suggest that the T. maritima UDG is a member of a new class of DNA repair enzymes.     

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.     

X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG.

Uracil-DNA glycosylase (UDG), a key highly conserved DNA repair enzyme involved in uracil excision repair, was discovered in Escherichia coli . The Bacillus subtilis bacteriophage, PBS-1 and PBS-2, which contain dUMP residues in their DNA, express a UDG inhibitor protein, Ugi which binds to UDG very tightly to form a physiologically irreversible complex. The X-ray analysis of the E. coli UDG ( Ec UDG)-Ugi complex at 3.2 A resolution, leads to the first structure elucidation of a bacterial UDG molecule. This structure is similar to the enzymes from human and viral sources. A comparison of the available structures involving UDG permits the delineation of the constant and the variable regions of the molecule. Structural comparison and mutational analysis also indicate that the mode of action of the enzyme from these sources are the same. The crystal structure shows a remarkable spatial conservation of the active site residues involved in DNA binding in spite of significant differences in the structure of the enzyme-inhibitor complex, in comparison with those from the mammalian and viral sources. Ec UDG could serve as a prototype for UDGs from pathogenic prokaryotes, and provide a framework for possible drug development against such pathogens with emphasis on features of the molecule that differ from those in the human enzyme.     

Contrasting effects of mutating active site residues, aspartic acid 64 and histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein

Uracil, a promutagenic base, arises in DNA by spontaneous deamination of cytosine or by the malfunctioning of DNA polymerases. To maintain the genomic integrity, cells possess a highly conserved base excision repair enzyme, uracil-DNA glycosylase (UDG). UDGs have a notably high turnover number and strict specificity for uracil in DNA. UDGs are inhibited by a small proteinaceous inhibitor, Ugi, which acts as a transition state substrate mimic. Crystal structure studies have identified the residues crucial in catalysis, and in their interaction with Ugi. Here, we report on the mutational analyses of D64 (D64H and D64N) and H187 (H187C, H187L and H187R) in the active site pocket of Escherichia coli UDG. The mutants were compromised in uracil excision by approximately 200-25,000 fold when compared to the native protein. In contrast, our analysis of the in vivo formed UDG-Ugi complexes on urea gels shows that D64 and H187 contribute minimally to the interaction of the two proteins. Thus, our findings provide further evidence to the primary function of D64 and H187 in catalysis.     

Uracil-DNA glycosylase in the extreme thermophile Archaeoglobus fulgidus

Uracil-DNA glycosylase (UDG) is an essential enzyme for maintaining genomic integrity. Here we describe a UDG from the extreme thermophile Archaeoglobus fulgidus. The enzyme is a member of a new class of enzymes found in prokaryotes that is distinct from the UDG enzyme found in Escherichia coli, eukaryotes, and DNA-containing viruses. The A. fulgidus UDG is extremely thermostable, maintaining full activity after heating for 1.5 h at 95 degrees C. The protein is capable of removing uracil from double-stranded DNA containing either a U/A or U/G base pair as well as from single-stranded DNA. This enzyme is product-inhibited by both uracil and apurinic/apyrimidinic sites. The A. fulgidus UDG has a high degree of similarity at the primary amino acid sequence level to the enzyme found in Thermotoga maritima, a thermophilic eubacteria, and suggests a conserved mechanism of UDG-initiated base excision repair in archaea and thermophilic eubacteria.     

Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases

Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.     

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.     

Contribution of a conserved phenylalanine residue to the activity of Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase (UDG) excises uracil from DNA to initiate repair of this lesion. This important DNA repair enzyme is conserved in viruses, bacteria, and eukaryotes. One residue that is conserved among all the members of the UDG family is a phenylalanine that stacks with uracil when it is flipped out of the DNA helix into the enzyme active site. To determine what contribution this conserved Phe residue makes to the activity of UDG, Phe-77 in the Escherichia coli enzyme was mutated to three different amino acid residues, alanine (UDG-F77A), asparagine (UDG-F77N), and tyrosine (UDG-F77Y). The effects of these mutations were measured on the steady-state and pre-steady-state kinetics of uracil excision in addition to enzyme.DNA binding kinetics. The overall excision activity of each of the mutants was reduced relative to the wild-type enzyme; however, each mutation gave rise to a different kinetic phenotype with different effects on substrate binding and catalysis. The excision activity of UDG-F77N was the most severely compromised, but this enzyme still bound to uracil-containing DNA at about the same rate as wild-type UDG. In contrast, the decrease in the excision activity of UDG-F77A is likely to reflect a greater reduction in uracil-DNA binding than in the catalytic step. Overall, the effects of the mutations on catalysis are best correlated with the polarity of the substituted residue such that an increase in polarity decreases the efficiency of uracil excision.     

Vaccinia virus uracil DNA glycosylase interacts with the A20 protein to form a heterodimeric processivity factor for the viral DNA polymerase

The vaccinia virus E9 protein, the catalytic subunit of the DNA polymerase holoenzyme, is inherently distributive under physiological conditions, although infected cells contain a highly processive form of the enzyme. The viral A20 protein was previously characterized as a stoichiometric component of the processivity factor, and an interaction between A20 and E9 was documented in vivo. A20 has been shown to interact with D4, the virally encoded uracil DNA glycosylase (UDG), by yeast-two hybrid and in vitro analysis. Here we confirm that UDG and A20 interact in vivo and show that temperature-sensitive viruses with lesions in the D4R gene show a profound defect in DNA synthesis at the non-permissive temperature. Moreover, cytoplasmic extracts prepared from these infections lack processive polymerase activity in vitro, implicating D4 in the assembly or activity of the processive polymerase. Upon overexpression of 3xFLAG-UDG, A20, and E9 in various combinations, we purified dimeric and trimeric UDG-A20 and UDG-A20-polymerase complexes, respectively. These complexes are stable in 750 mm NaCl and can be further purified by Mono Q chromatography. Notably, the trimeric complex displays robust processive polymerase activity, and the dimeric complex can confer processivity on purified E9. Consistent with previous reports that the catalytic activity of UDG is dispensable for virus replication in tissue culture, we find that the role of UDG role in the polymerase complex is not diminished by mutations targeting residues involved in uracil recognition or excision. Our cumulative data support the conclusion that A20 and UDG form a heterodimeric processivity factor that associates with E9 to comprise the processive polymerase holoenzyme.     

A uracil-DNA glycosylase inhibitor encoded by a non-uracil containing viral DNA

Uracil-DNA glycosylase (UDG) is an enzyme involved in the base excision repair pathway. It specifically removes uracil from both single-stranded and double-stranded DNA. The genome of the Bacillus subtilis phage 29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5'-end. Replication of 29 DNA starts by a protein-priming mechanism and generates intermediates that have long stretches of single-stranded DNA. By using in vivo chemical cross-linking and affinity chromatography techniques, we found that UDG is a cellular target for the early viral protein p56. Addition of purified protein p56 to B. subtilis extracts inhibited the endogenous UDG activity. Moreover, extracts from 29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by 29 to prevent the action of the base excision repair pathway if uracil residues arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a non-uracil-containing viral DNA.     

Substrate specificities and functional characterization of a thermo-tolerant uracil DNA glycosylase (UdgB) from Mycobacterium tuberculosis

Uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base (uracil) excision repair pathway. Ung, a highly conserved protein, is the only UDG characterized so far in mycobacteria. Here, we show that Rv1259 from Mycobacterium tuberculosis codes for a double-stranded DNA (dsDNA) specific UDG (MtuUdgB). MtuUdgB is thermo-tolerant, contains Fe-S cluster and, in addition to uracil, it excises ethenocytosine and hypoxanthine from dsDNA. MtuUdgB is product inhibited by AP-site containing dsDNA but not by uracil. While MtuUdgB excises uracil present as a single-nucleotide bulge in dsDNA, it is insensitive to inhibition by dsDNA containing AP-site in the bulge. Interestingly, in the presence of cellular factors, the uracil excision activity of MtuUdgB is enhanced, and when introduced into E. coli (ung(-)), it rescues its mutator phenotype and prevents C to T mutations in DNA. Novel features of the mechanism of action of MtuUdgB and the physiological significance of the family 5 UDG in mycobacteria have been discussed.     

Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung

Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes.     

Crystal structure of a family 4 uracil-DNA glycosylase from Thermus thermophilus HB8

Uracil-DNA glycosylase (UDG; EC 3.2.2.-) removes uracil from DNA to initiate DNA base excision repair. Since hydrolytic deamination of cytosine to uracil is one of the most frequent DNA-damaging events in all cells, UDG is an essential enzyme for maintaining the integrity of genomic information. For the first time, we report the crystal structure of a family 4 UDG from Thermus thermophilus HB8 (TthUDG) complexed with uracil, solved at 1.5 angstroms resolution. As opposed to UDG enzymes in its other families, TthUDG possesses a [4Fe-4S] cluster. This iron-sulfur cluster, which is distant from the active site, interacts with loop structures and has been suggested to be unessential to the activity but necessary for stabilizing the loop structures. In addition to the iron-sulfur cluster, salt-bridges and ion pairs on the molecular surface and the presence of proline on loops and turns is thought to contribute to the enzyme's thermostability. Despite very low levels of sequence identity with Escherichia coli and human UDGs (family 1) and E.coli G:T/U mismatch-specific DNA glycosylase (MUG) (family 2), the topology and order of secondary structures of TthUDG are similar to those of these distant relatives. Furthermore, the coordinates of the core structure formed by beta-strands are almost the same. Positive charge is distributed over the active-site groove, where TthUDG would bind DNA strands, as do UDG enzymes in other families. TthUDG recognizes uracil specifically in the same manner as does human UDG (family 1), rather than guanine in the complementary strand DNA, as does E.coli MUG (family 2). These results suggest that the mechanism by which family 4 UDGs remove uracils from DNA is similar to that of family 1 enzymes.     

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ?29 DNA mimic protein p56

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.     

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.     

Characterization of Bacillus subtilis uracil-DNA glycosylase and its inhibition by phage φ29 protein p56

Uracil-DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram-positive organism. The purified enzyme removes uracil preferentially from single-stranded DNA over double-stranded DNA, exhibiting higher preference for U:G than U:A mismatches. Furthermore, we have identified key amino acids necessary for B. subtilis UDG activity. Our results showed that Asp-65 and His-187 are catalytic residues involved in glycosidic bond cleavage, whereas Phe-78 would participate in DNA recognition. Recently, it has been reported that B. subtilis phage φ29 encodes an inhibitor of the UDG enzyme, named protein p56, whose role has been proposed to ensure an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracils present in the φ29 genome. In this work, we also show that a φ29-related phage, GA-1, encodes a p56-like protein with UDG inhibition activity. In addition, mutagenesis analysis revealed that residue Phe-191 of B. subtilis UDG is critical for the interaction with φ29 and GA-1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition.