Optimisation of the surface electrostatics as a strategy for cold adaptation of uracil-DNA N-glycosylase (UNG) from Atlantic cod (Gadus morhua)

Cold-adapted enzymes are characterised by an increased catalytic efficiency and reduced temperature stability compared to their mesophilic counterparts. Lately, it has been suggested that an optimisation of the electrostatic surface potential is a strategy for cold adaptation for some enzymes. A visualisation of the electrostatic surface potential of cold-adapted uracil-DNA N-glycosylase (cUNG) from Atlantic cod indicates a more positively charged surface near the active site compared to human UNG (hUNG). In order to investigate the importance of the altered surface potential for the cold-adapted features of cod UNG, six mutants have been characterised and compared to cUNG and hUNG. The cUNG quadruple mutant (V171E, K185V, H250Q and H275Y) and four corresponding single mutants all comprise substitutions of residues present in the human enzyme. A human UNG mutant, E171V, comprises the equivalent residue found in cod UNG. In addition, crystal structures of the single mutants V171E and E171V have been determined. Results from the study show that a more negative electrostatic surface potential reduces the activity and increases the stability of cod UNG, and suggest an optimisation of the surface potential as a strategy for cold-adaptation of this enzyme. Val171 in cod UNG is especially important in this respect.     

Identification,cloning, and expression of uracil-DNA glycosylase from Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>): characterization and homology modeling of the cold-active catalytic domain

Two distinct forms of the highly conserved uracil-DNA glycosylase (UNG) have been isolated from Atlantic cod (Gadus morhua) liver cDNA by rapid amplification of cDNA ends (RACE). From the cDNA sequences, both forms were deduced to encode an open reading frame of 301 amino acids, with an identical 267-amino-acid C-terminal region and different N-terminal regions of 34 amino acids. By comparison with the human UNG sequences, the two forms were identified as possible mitochondrial (cUNG1) and nuclear (cUNG2) forms. Several constructs of recombinant cUNG (rcUNG) were expressed in Escherichia coli in order to optimize the yield. The recombinant enzyme was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity and stability experiments showed that rcUNG was similar to cUNG previously purified from Atlantic cod liver, and was more pH- and temperature labile than a recombinant human UNG (rhUNG). Under optimal assay conditions for both rcUNG and rhUNG, the turnover number (k(cat)) was three times higher for rcUNG compared with rhUNG, with an identical K(M), resulting in a threefold higher catalytic efficiency (k(cat)/K(M)) for rcUNG. These activity and stability experiments reveal cold-adapted features in rcUNG. Homology models of the catalytic domains of Atlantic cod (cUNG) and mouse uracil-DNA glycosylase (mUNG) were built using the human UNG (hUNG) crystal structure as a template. The unique amino acid substitutions observed in cod UNG were mainly located in the N- and C-terminal parts of the sequence. The analysis indicated a more stable N-terminal, a more flexible C-terminal, and a less stabilized core in cUNG as compared with the mammalian UNGs. Substitution of several amino acids in or near the DNA-binding site in cUNG could give rise to a more positively charged surface and a higher electrostatic potential near the active site compared with the mammalian UNGs. The higher potential may increase the electrostatic interactions between the enzyme and DNA, and may explain the increased substrate affinity and, in combination with the higher flexibility, the higher catalytic efficiency observed for rcUNG.     

Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua)

Uracil-DNA glycosylase (UDG; UNG) has been purified 17000-fold from Atlantic cod liver (Gadus morhua). The enzyme has an apparent molecular mass of 25 kDa, as determined by gel filtration, and an isoelectric point above 9.0. Atlantic cUNG is inhibited by the specific UNG inhibitor (Ugi) from the Bacillus subtilis bacteriophage (PBS2), and has a 2-fold higher activity for single-stranded DNA than for double-stranded DNA. cUNG has an optimum activity between pH 7.0-9.0 and 25-50 mM NaCl, and a temperature optimum of 41 degrees C. Cod UNG was compared with the recombinant human UNG (rhUNG), and was found to have slightly higher relative activity at low temperatures compared with their respective optimum temperatures. Cod UNG is also more pH- and temperature labile than rhUNG. At pH 10.0, the recombinant human UNG had 66% residual activity compared with only 0.4% for the Atlantic cUNG. At 50 degrees C, cUNG had a half-life of 0.5 min compared with 8 min for the rhUNG. These activity and stability experiments reveal cold-adapted features in cUNG.     

A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.     

Characterization of human cytomegalovirus uracil DNA glycosylase (UL114) and its interaction with polymerase processivity factor (UL44)

Here, we report the molecular characterization of the human cytomegalovirus uracil DNA glycosylase (UNG) UL114. Purified UL114 was shown to be a DNA glycosylase, which removes uracil from double-stranded and single-stranded DNA. However, kinetic analysis has shown that viral UNG removed uracil more slowly compared with the core form of human UNG (Δ84hUNG), which has a catalytic efficiency (k_cat/K_M) 350- to 650-fold higher than that of UL114. Furthermore, UL114 showed a maximum level of DNA glycosylase activity at equimolar concentrations of the viral polymerase processivity factor UL44. Next, UL114 was coprecipitated with DNA immobilized to magnetic beads only in the presence of UL44, suggesting that UL44 facilitated the loading of UL114 on DNA. Moreover, mutant analysis demonstrated that the C-terminal part of UL44 (residues 291-433) is important for the interplay with UL114. Immunofluorescence microscopy revealed that UL44 and UL114 colocalized in numerous small punctuate foci at the immediate-early (5 and 8 hpi) phases of infection and that these foci grew in size throughout the infection. Furthermore, coimmunoprecipitation assays with cellular extracts of infected cells confirmed that UL44 associated with UL114. Finally, the nuclear concentration of UL114 was estimated to be 5- to 10-fold higher than that of UL44 in infected cells, which indicated a UL44-independent role of UL114. In summary, our data have demonstrated a catalytically inefficient viral UNG that was highly enriched in viral replication foci, thus supporting an important role of UL114 in replication rather than repair of the viral genome.     

Electrostatic interactions play an essential role in DNA repair and cold-adaptation of Uracil DNA glycosylase

Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k_cat and reduced K_m relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K_m) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation. Figure Electrostatic isosurfaces of cod uracil DNA glycosylase in complex with double stranded DNA     

尿嘧啶N糖基化酶(UNG)的研究进展

尿嘧啶N糖基化酶是碱基切除修复过程中的重要组分。本文从酶的概况、在生物体内的分布范围、人尿嘧啶N糖基化酶的基因结构、基因表达与调控、酶的作用机制等方面进行了介绍,并讨论了进一步研究的意义与方向。     

Analysis of uracil DNA glycosylase (UNG2) stimulation by replication protein A (RPA) at ssDNA-dsDNA junctions

Replication Protein A (RPA) is a single-stranded DNA binding protein that interacts with DNA repair proteins including Uracil DNA Glycosylase (UNG2). Here, I report DNA binding and activity assays using purified recombinant RPA and UNG2. Using synthetic DNA substrates, RPA was found to promote UNG2's interaction with ssDNA-dsDNA junctions regardless of the DNA strand polarity surrounding the junction. RPA stimulated UNG2's removal of uracil bases paired with adenine or guanine in DNA as much as 17-fold when the uracil was positioned 21 bps from ssDNA-dsDNA junctions, and the largest degree of UNG2 stimulation occurred when RPA was in molar excess compared to DNA. I found that RPA becomes sequestered on ssDNA regions surrounding junctions which promotes its spatial targeting of UNG2 near the junction. However, when RPA concentration exceeds free ssDNA, RPA promotes UNG2's activity without spatial constraints in dsDNA regions. These effects of RPA on UNG2 were found to be mediated primarily by interactions between RPA's winged-helix domain and UNG2's N-terminal domain, but when the winged-helix domain is unavailable, a secondary interaction between UNG2's N-terminal domain and RPA can occur. This work supports a widespread role for RPA in stimulating uracil base excision repair.     

New insights on the role of the gamma-herpesvirus uracil-DNA glycosylase leucine loop revealed by the structure of the Epstein-Barr virus enzyme in complex with an inhibitor protein

Epstein-Barr virus (EBV) is a human gamma-herpesvirus. Within its 86 open reading frame containing genome, two enzymes avoiding uracil incorporation into DNA can be found: uracil triphosphate hydrolase and uracil-DNA glycosylase (UNG). The latter one excises uracil bases that are due to cytosine deamination or uracil misincorporation from double-stranded DNA substrates. The EBV enzyme belongs to family 1 UNGs. We solved the three-dimensional structure of EBV UNG in complex with the uracil-DNA glycosylase inhibitor protein (Ugi) from bacteriophage PBS-2 at a resolution of 2.3 A by X-ray crystallography. The structure of EBV UNG encoded by the BKRF3 reading frame shows the excellent global structural conservation within the solved examples of family 1 enzymes. Four out of the five catalytic motifs are completely conserved, whereas the fifth one, the leucine loop, carries a seven residue insertion. Despite this insertion, catalytic constants of EBV UNG are similar to those of other UNGs. Modelling of the EBV UNG-DNA complex shows that the longer leucine loop still contacts DNA and is likely to fulfil its role of DNA binding and deformation differently than the enzymes with previously solved structures. We could show that despite the evolutionary distance of EBV UNG from the natural host protein, bacteriophage Ugi binds with an inhibitory constant of 8 nM to UNG. This is due to an excellent specificity of Ugi for conserved elements of UNG, four of them corresponding to catalytic motifs and a fifth one corresponding to an important beta-turn structuring the catalytic site.     

Mimicking damaged DNA with a small molecule inhibitor of human UNG2

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.     

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).     

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.     

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl(2). The functional significance of the UNG2.PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-(3)H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl(2). The stimulatory effect of PCNA was increased by the addition of MgCl(2); however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl(2) and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.     

Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase

Leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that catalyzes leucylation of tRNA(Leu). Several mutants in the CP1 domain of Escherichia coli LeuRS were obtained by introduction of restriction endonuclease sites into its gene, leuS. Of these mutants, only LeuRS-A293F had decreased activity (46%) compared to the native enzyme. To investigate the effect of A293 on enzyme function, A293 was mutated to Y, G, I, R, or D. The mutants were impaired in activity and editing function to varying extents. The decrease in K(m) values for three substrates showed that the binding of ATP to these mutants became much stronger. The inhibition of ATP binding to most of the mutants was also stronger. In particular, LeuRS-A293D had the lowest activity, the strongest ATP binding, and the most impaired editing function. A red shift of the fluorescence emission maximum of LeuRS-A293D indicated a less hydrophobic chromophore environment and a relatively more flexible dynamic conformation. The change in T(m) of LeuRS-A293D was higher than that of all other substitutions. Evidence from sequence alignment and crystal structure of LeuRS from Thermus thermophilus shows that A293 was conserved as R (K) or A and is located at a small helix in the editing domain of the enzyme facing the active site. Hence, any amino acid substitution of A293 may affect the stability of the helix, which may lead to impaired editing function and aminoacylation activity and may be indirectly involved in ATP binding.     

The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover

Background

The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.

Methodology/Principal Findings

We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.

Conclusions/Significance

This work identifies UNG2 and SMUG1 as novel targets for CRL4^DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4^DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4^DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.     

Human ribosomal protein S3 (hRpS3) interacts with uracil-DNA glycosylase (hUNG) and stimulates its glycosylase activity

Human ribosomal protein S3 (hRpS3) is a small ribosomal subunit showing apurinic/apyrimidinic (AP) lyase activity and has been suggested to play a role in the cellular DNA-damage response pathway. However, the functional interactions between hRpS3 and other base excision repair (BER) DNA glycosylases have not been reported. We identified, for the first time, the interaction between hRpS3 and human uracil-DNA glycosylase (hUNG) and investigated the functional consequences of this interaction. hRpS3 was shown to interact with hUNG in co-immunoprecipitation assay using transiently transfected HEK293 cells and GST pull-down assay using microbial expression systems. In an assay using a 5'-end-radiolabeled 39-mer oligonucleotide duplex containing a U/G mismatch, hRpS3 dramatically stimulated the uracil-excision activity of hUNG, whereas hRpS3 alone had no cleavage activity. Pre-incubation of hRpS3 with the U/G mismatch containing DNA duplex also increased the hUNG uracil-excision activity; however, hRpS3 did not increase the DNA binding activity of hUNG in a trapping assay of hUNG and the U/G mismatch containing DNA duplex using UV cross-linking. hRpS3 has been suggested to stimulate the uracil-excision activity of hUNG by enhancing its dissociation from AP sites and increasing its turn-over rate. The disruption of hRpS3 by small-interfering RNA (siRNA-hRpS3) transfection reduced the uracil-excision activity preserved in cell extracts, whereas the supplement of purified hRpS3 retained uracil-excision activity. These results strongly suggest that hRpS3 may be involved in the uracil-excision pathway, probably by participating in the DNA repair mechanism to remove uracil generated by the deamination of cytosine in DNA, and by preventing C/G-->T/A transition mutations.     

Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms

DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.