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Human mitochondrial uracil-DNA glycosylase preform (UNG1) is processed to two forms one of which is resistant to inhibition by AP sites
Introduction
Materials And Methods
Materials
Plasmid constructions, co-transfection and generation of recombinant virus
Purification of recombinant UNG
Subcellular fractionation of HeLa cells
Assays
Proteolytic cleavage of UNG
Results
Expression of human UNG in insect cells
Purification of UNG1 from insect cells
Human mitochondria contain two processed forms of UNG1, one of which is not formed by MPP
Catalytic properties of UNG1[Delta]29
UNG1[Delta]29 is not product inhibited by AP sites
UNG1[Delta]29 and UNG1[Delta]77 are stimulated by HAP1
Discussion
Acknowledgements
References
Human mitochondrial uracil-DNA glycosylase preform (UNG1) is processed to two forms one of which is resistant to inhibition by AP sites
ABSTRACT
INTRODUCTION
Uracil-DNA glycosylases (UDG) catalyse the first step in the base excision repair (BER) pathway for removal of uracil from DNA. Uracil in DNA results from either deamination of cytosine, a premutagenic lesion (1), or incorporation of dUMP instead of dTMP during replication (2). Genes or cDNA sequences for UDGs from different organisms predict sizes ranging from 204 to 359 amino acid residues. Crystal structures of the HSV-1 (3) and the human (4) C-terminal regions of ~220 residues are very similar and contain both the DNA-binding and catalytic domains (5). Catalysis proceeds via the insertion of a conserved leucine into the DNA minor groove following expulsion of uracil into the buried catalytic pocket, concomitant with compression of flanking DNA phosphates and surrounding amino acid residues to form a productive complex (5). N-terminal sequences of UDGs are not required for catalytic activity and vary widely both in length and amino acid composition. They are considerably longer in the eukaryotic and herpesviral UDGs as compared with the bacterial, mycoplasma and poxviral UDGs, in agreement with their involvement in subcellular localization in mammalian cells (6,7). The human UNG gene was the first mammalian gene demonstrated to encode both nuclear (UNG2) and mitochondrial (UNG1) isoforms of an enzyme (6), by a mechanism involving transcription from two different UNG promoters and alternative splicing (7). The mRNAs for UNG1 and UNG2 encode 35 and 44 unique N-terminal residues that are required for mitochondrial and nuclear translocation, respectively, whereas the 269 residues downstream of the unique sequences are common to the two isoforms (7).
Purification of UNG proteins from various cells and subcellular fractions have resulted in active enzyme species that differ in size as well as in some biochemical properties (6,8-11). Recently, purification of human UNG protein from HeLa cells in the presence of a cocktail of protease inhibitors resulted in an enzyme species of 35-37 kDa, which is close to the size predicted from the open reading frame for UNG2 and UNG1 mRNAs (12). A part of the N-terminal region in UNG located C-terminal to the unique sequences has been demonstrated to bind replication protein A (13). Thus, N-terminal regions in UNG proteins in addition to having a role in subcellular targeting may also have functions that are not yet elucidated. In the present work we present evidence that the mitochondrial preprotein UNG1 is processed to yield two catalytically active forms of 31 and 26 kDa, respectively. In addition we show that the 31 kDa form has certain unique properties not described for other uracil-DNA glycosylases, most notably the resistance to the inhibitory effect of AP sites observed with other UDGs.
MATERIALS AND METHODS
Materials
The BacPAK Baculovirus Expression System kit was from Clontech Laboratories (Palo Alto, CA). Proteinase K, RNase A and SeaPlaque agarose were from Sigma (St Louis, MO). The protease inhibitors and DIG glycan/protein double labelling kit were from Boehringer-Mannheim (Germany). Anti-phosphoprotein antibodies were from Zymed Laboratories (San Francisco, CA). Restriction enzymes, DNA-modifying enzymes and linkers were from New England Biolabs (Beverly, MA) and Promega (Madison, WI). Media components for bacterial culture were from Difco (Detroit, MI) and media for insect cell culture were from Gibco BRL (Gaithersburg, MD). Chromatographic matrices were from Pharmacia Biotech (Uppsala, Sweden).
Plasmid constructions, co-transfection and generation of recombinant virus
In brief, the pSELECT system (Promega) was used to introduce an NdeI site at the ATG start codon (position 110) in UNG15 (14), containing the complete UNG1 open reading frame. After blunting of the NdeI site, XhoI linkers were added directly upstream of the ATG codon. The XhoI-HpaI fragment of UNG15 containing its stop signal was cloned into the XhoI and SmaI sites in the polylinker of the pBacPAC8 baculovirus expression vector (Clontech). The resultant construct was named pBacUNG1 (6.5 kb) and correct in-frame sequence was confirmed by DNA sequencing.
Spodoptera frugiperda Sf21 cells were cultured and infected with recombinant virus essentially as described (15). Lipofectin-mediated co-transfection of pBacUNG and AcMNPV or BacPAK6 viral DNA into Sf21 cells was performed according to the manufacturer's protocol (Clontech). The recombinant viruses were isolated 72 h post-infection by several rounds of plaque purification (15) and screened by activity measurements and Southern analysis using an UNG-specific probe. The recombinant virus BacUNG10 was selected for scale-up experiments.
Purification of recombinant UNG
Sf21 cells were cultured in suspension cultures, infected with the recombinant baculovirus at a multiplicity of infection of 20 and harvested 36 h post-infection. All subsequent steps were performed at 4°C. The frozen cell pellet (62 g) from 7 l culture was thawed and resuspended in 200 ml of lysis buffer containing 50 mM Tris-acetate (pH 7), 10 mM NaCl, 1 mM DTT and protease inhibitors (all buffers used during protein purification were degassed and contained 0.5 mM PMSF, 1 µg/ml each of pepstatin A and leupeptin and 2 µg/ml of aprotinin). Cells were lysed by sonication and complete cell disintegration was verified by phase-contrast microscopy. UNG proteins were purified essentially as described (16) except that nucleic acid precipitation using protamine sulphate was omitted. Fractions from each chromatographic step were analysed by SDS-PAGE and western analysis using the polyclonal antibody PU101 directed against the catalytic 26 kDa domain (16). Purified proteins were thoroughly dialysed against 50 mM NH4HCO3 and N-terminally sequenced using an Applied Biosystems 477A Sequenator.
Subcellular fractionation of HeLa cells
In brief, 2 × 107 logarithmically growing HeLa cells were harvested by scraping and washed twice in ice-cold PBS, resuspended in 5 ml hypotonic buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1.5 mM MgCl2) and lysed by 15 strokes in a 7 ml Dounce homogenizer. Nuclei were pelleted by centrifugation at 500 g for 2 min and further purified in a 30/35% OptiPrep (Nycomed Pharma, Norway) step gradient according to the manufacturer's recommendations. Finally, the nuclei were incubated in 0.5 ml PBS, 0.2% Triton X-100 for 30 min on ice, adjusted to a final protein concentration of 1 mg/ml in the same buffer, snap frozen in liquid N2 and stored at -70°C. For fractionation of mitochondria, 108 HeLa cells were harvested as above, resuspended in 6 ml diluent B (8% sucrose, 1 mM EDTA, 20 mM Tricine-NaOH, pH 7.8) and lysed by 30 strokes in a 7 ml Dounce homogenizer. The homogenate was centrifuged at 1000 g for 10 min, the pellet resuspended in 5 ml diluent B and recentrifuged as above and the two post-nuclear supernatants (PNS) combined. The PNS was further centrifuged at 3000 g for 10 min to pellet the heavy mitochondrial fraction (HMF) and the supernatant recentrifuged at 17 000 g for 15 min to pellet the light mitochondrial fraction (LMF). The LMF was further fractionated in an OptiPrep continuous density gradient (starting concentration 17%) at 270 000 g for 3 h. All fractions, including the LMF supernatant (cytosol/microsomal fraction) were immediately snap frozen in liquid N2 and stored at -70°C.
Assays
Unless otherwise specified, UDG activity was measured in 20 µl of assay mixture under the standard conditions essentially as described (16). BioRad protein assay was used to measure protein concentrations using BSA as standard. Km, Vmax and uracil inhibition were determined in the presence of 0.5-10 µM [3H]dUMP-containing DNA (concentration referring to uracil). Single-stranded (ss) substrate was prepared by heating the double-stranded (ds) substrate at 100°C for 10 min and then immediately cooling on ice. The concentrations of the enzyme protein were 0.1 and 0.3 ng/20 µl assay mixture for ssDNA and dsDNA, respectively, and the NaCl concentrations were 10, 30 and 60 mM. Inhibition by uracil was studied in the presence of 2 and 5 mM uracil at each of the above NaCl concentrations. Marker enzymes for the following organelles were assayed essentially as described; cytochrome c oxidase (mitochondrial marker) (8), acid phosphatase (lysosomes) (17) and catalase (peroxisomes) (18). Sequence specificity of uracil excision was assayed essentially as described (19) and binding of UNG to ssDNA or dsDNA matrices were as described (4) except that larger matrix volumes (0.5 × 5 cm) were used. Post-translational glycosylation and phosphorylation were assayed using a DIG glycan/protein double labelling kit and anti-phosphoprotein antibodies, respectively, according to the recommended protocols. The effect of HAP1 (a gift from I. D. Hickson, Oxford, UK) on uracil excision was assayed by including varying concentrations of HAP1 (0-250 times molar excess with respect to UNG) in standard UDG assays. For a time curve experiment, similar assays were performed for specific lengths of time (0-60 min) using UNG only (50 pmol) or UNG (50 pmol) and a 25× molar excess of HAP1. The effect of AP sites was studied by including the different specified concentrations of double-stranded oligonucleotides containing AP sites [5[prime]-TGAAATTG(AP)TATCCGCTCA-3[prime] opposite either AP:A or AP:G] in the standard UDG assay.
Proteolytic cleavage of UNG
Aliquots of 50 ng UNG were treated with different primary HeLa subcellular fractions (4 µg total protein each) and UDG activity was assayed after the indicated time points. An equal volume of SDS loading buffer was added to the reaction mixtures for subsequent western analysis. Cleavage by secondary subcellular fractions (from the light mitochondrial primary fraction) was as above except that 2 µg total protein were added. To analyse cleavage by proteinase K, 7 pmol of UNG was incubated with or without 0.2 µg proteinase K in a total volume of 100 µl UDG assay buffer at room temperature. Reactions were stopped by addition of 100 µl phenylmethylsulphonylfluoride (PMSF; 2 mM in UDG buffer), immediately cooled on ice and subjected to western analysis and UDG assay.
Figure 1. Optimization of UNG expression in Sf21 cells after infection with BacUNG10. (A) Western analysis of cells harvested at different time points after infection. The cells were directly lysed in SDS loading buffer and boiled for 5 min to avoid proteolytic cleavage after cell disruption. (B) Specific UDG activity in non-denatured cell-free extracts from the same samples as in (A). Infection of Sf21 insect cells with BacUNG10 resulted in a new protein that was easily detected 24 h post-infection when cells were directly lysed in denaturing buffer and subjected to SDS-PAGE and western analysis (Fig. Figure 2. SDS-PAGE analysis after different steps in the purification of recombinant UNG from the insect cells. Lane 1, 1 µg of protein in crude extract; lane 2, 0.5 µg of protein after DE52/CM Sephadex chromatography; lane 3, 0.3 µg of protein after Superdex 75 gel filtration chromatography; lane 4, 0.3 µg of protein after MonoS chromatography. The molecular weight standards used were phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.3 kDa). In an attempt to purify UNG1-derived proteins, a 7 l suspension culture of baculovirus-infected Sf21 cells was harvested 36 h post-infection. Western analysis demonstrated that only trace amounts of the putative full-length protein were present after lysis in non-denaturing buffer (data not shown). Addition of a protease inhibitor cocktail containing PMSF, pepstatin A, leupeptin and aprotinin prior to cell homogenization did not improve the yield of full-length UNG1, supporting that proteolysis occurs in vivo. Samples from the various purification steps were analysed by SDS-PAGE and silver staining (Fig. Figure 3. N-terminal presequences of UNG1 and UNG2. Arrows indicate the cleavage sites for UNG1[Delta]29, UNG1[Delta]75 and UNG1[Delta]77. The numbers below indicate the position of amino acids from the UNG1 N-terminus. Bold letters and asterisks indicate basic residues at positions observed in known substrates for MPP. Underlined sequences represent the common sequence for UNG1 and UNG2. To investigate whether the observed UNG1 processing in insect cells also occurs in human cells, HeLa mitochondria were isolated by OptiPrep density gradient centrifugation and subjected to SDS-PAGE and western analysis using a polyclonal antibody directed towards the common catalytic domain of UNG1 and UNG2. As shown in Figure Figure 4. Western analysis of mitochondrial HeLa extracts demonstrating the presence of endogenous 26 and 31 kDa species. Lane 1, 0.05 ng UNG1[Delta]29 (31 kDa) partially cleaved by HeLa mitochondrial extract to 26 kDa UNG1; lane 2, 0.5 µg total protein from refractionated light mitochondrial fraction, directly lysed in SDS loading buffer; lane 3, as lane 2, but mitochondria incubated at 37°C for 1 h before lysis; lane 4, 0.025 ng each of purified UNG1[Delta]29 and UNG1[Delta]77. To investigate whether the cleavage factor(s) was specific for mitochondria, various HeLa subcellular fractions were isolated and purified recombinant UNG1[Delta]29 incubated in the presence of the various fractions. A major fraction of UNG1[Delta]29 was cleaved by incubation with HeLa cell mitochondrial extracts, yielding a 26 kDa UNG species (Fig. Figure 5. (A) Western analysis showing cleavage of UNG1[Delta]29 by different primary subcellular fractions without incubation and after 60 min incubation at 37°C. (B) (upper) Western analysis of purified UNG1[Delta]29 incubated in the presence of various fractions after refractionation of the light mitochondrial fraction; (lower) cytochrome c oxidase activity (filled circles) and endogenous UDG activity (filled triangles) in individual fractions. Density gradient fractionation of the LMF demonstrated that the UNG1[Delta]29 cleaving activity and the cytochrome c oxidase activity co-eluted. The same fractions also contained the major fraction of endogeneous UNG activity in the LMF (Fig. Figure 6. Influence of divalent cations and protease inhibitors on UNG1[Delta]29 cleavage. (A) UNG1[Delta]29 was incubated with light mitochondrial fraction 14 (Fig. 5) in the presence of EDTA or MnCl2 (given as final concentrations). Lane 1, unincubated UNG1[Delta]29 control with 10 mM EDTA; lane 2, 10 mM MnCl2; lane 3, no EDTA or MnCl2 added; lane 4, 5 mM EDTA; lane 5, 10 mM EDTA. Fractions in lanes 2-5 were incubated for 1 h at 37°C. (B) Western analysis showing cleavage of UNG1[Delta]29 by the light mitochondrial fraction 14 in the presence of various protease inhibitors and after boiling of the light mitochondrial fraction. Lane 1, UNG1[Delta]29 - LMF; lane 2, UNG1[Delta]29 + LMF; lane 3, UNG1[Delta]29 + LMF (boiled for 5 min prior to incubation); lanes 4-16, UNG1[Delta]29 + LMF + various protease inhibitors (given as final concentrations); lane 4, antipain dihydrochloride (50 µg/ml); lane 5, bestatin (40 µg/ml); lane 6, chymostatin (20 µg/ml); lane 7, E-64 (10 µg/ml); lane 8, leupeptin (1 µg/ml); lane 9, pepstatin (1 µg/ml); lane 10, phosphoramidon (20 µg/ml); lane 11, PefablockR (200 µg/ml), lane 12, Na2EDTA (200 µg/ml); lane 13, aprotinin (2 µg/ml); lane 14, CompleteR (1 mg/ml) ; lane 15, Complete, EDTA-freeR (1 mg/ml); lane 16, phenylmethylsulphonylfluoride (PMSF, 0.5 mM). An identical cleavage pattern was observed with the heavy mitochondrial fraction. The [Delta]29 cleavage site is entirely consistent with known substrate sites for the mitochondrial processing peptidase MPP, having an Arg at position -2 and -14 and a Lys at position -10 (20,21; Fig. To investigate the general susceptibility of UNG1[Delta]29 to proteolytic cleavage, the enzyme was subjected to proteinase K digestion (Fig. Figure 7. Effect of proteinase K treatment on UNG1[Delta]29 and UNG1[Delta]77. (A) Western analysis of UNG1[Delta]29 and UNG1[Delta]77 after proteinase K treatment for different lengths of time. (B) Change in the specific molar activities of the recombinant enzymes treated with proteinase K for different lengths of time. Empty squares, UNG1[Delta]29 only; filled squares, UNG1[Delta]29 + proteinase K; empty triangles, UNG1[Delta]77 only; filled triangles, UNG1[Delta]77 + proteinase K. Each value is the mean of three replicas with SEM values as indicated.
The presence of a 31 kDa UNG in human mitochondria resembling UNG1[Delta]29 and the fact that this species is enzymatically highly active justified further biochemical analysis of UNG1[Delta]29. The kinetic constants of UNG1[Delta]29 measured at different NaCl concentrations are given in Table 1. A similar salt optimum (60 mM) was observed for UNG1[Delta]29 as for UNG1[Delta]77 purified from human placenta (9), but kcat/Km of UNG1[Delta]29 was ~6-fold lower. Analysis of possible post-translational modifications in UNG1[Delta]29 demonstrated that the protein was neither glycosylated nor phosphorylated (data not shown). The specific activity of UNG1[Delta]29 using ssDNA as substrate was ~3-fold higher than that obtained with dsDNA substrate under standard assay conditions, similar to other UNG enzymes reported (9,16,22). Furthermore, the sequence context preference for uracil excision was essentially identical to that of UNG1[Delta]84 (16; data not shown). Table 1. The two products of the N-glycolytic reaction have been shown to be millimolar (uracil) and micromolar (AP sites) inhibitors of various UDGs, respectively (11,16,23,24). UNG1[Delta]29 showed a similar degree of inhibition by uracil (50% at 1.5 mM uracil). Surprisingly, however, no inhibition could be observed with oligonucleotides containing AP sites opposite either A or G (Fig. In addition to six unique N-terminal residues unique to UNG1, UNG1[Delta]29 contains the entire sequence common to UNG1 and UNG2 (Fig. Figure 8. (A) Effect of varying concentrations of AP site-containing oligonucleotides on UNG1[Delta]29 and UNG1[Delta]77. Either of the two different oligonucleotides containing AP sites (AP:G and AP:A) were used in the assay. Full (100%) activity of each of the two enzymes is measured in the absence of AP sites. Filled circles, UNG1[Delta]29 + AP:A; empty circles, UNG1[Delta]29 + AP:G; filled triangles, UNG1[Delta]77 + AP:A; empty triangles, UNG1[Delta]77 + AP:G. (B) Enhancement of UNG1[Delta]29 (shaded bars) and UNG1[Delta]77 (open bars) activity in the presence of varying concentrations of HAP1. Per cent increase is measured relative to the activity in the absence of HAP1. The values in both figures are the mean of three replicas with SEM values as indicated.
Using a baculovirus expression system, we have produced a 31 kDa recombinant form of human UNG1 (UNG1[Delta]29) that lacks 29 of the 35 N-terminal amino acids unique to UNG1, but which contains the complete common domain of UNG1 and UNG2. This form represents an enzymatically highly active protein that is apparently also present in human mitochondria in addition to the fully processed 26 kDa core catalytic domain. Cleavage resulting in the 26 kDa UNG1 takes place within the N-terminal sequence common to UNG1 and nuclear UNG2. This most likely explains the different results from various laboratories with regard to sizes and properties of UNG proteins purified from different mammalian cells, since leakage of mitochondrial proteases during cell fractionation could result in cleavage of UNG2, thus producing an enzyme species identical to the 26 kDa UNG1. Whether the 31 or the 26 kDa form represents the mature functional mitochondrial UNG remains unclear. Alternatively, both forms are present and serve distinct functions in mitochondrial uracil repair. Quantitatively, the 31 kDa form dominates both in the insect cells and isolated HeLa mitochondria, indicating that UNG1[Delta]29 is a functional mitochondrial enzyme. The presence of a putative amphiphilic helix at UNG1 positions 11-29, the cleavage immediately downstream of this region and the homology of the cleavage site to known MPP substrates furthermore suggest that UNG1 is translocated via the major mitochondrial import machinery (reviewed in 26) and cleaved by MPP to yield UNG1[Delta]29. This is also supported by the apparently effective in vivo formation of UNG1[Delta]29 in insect cells lacking endogenous UDG. Further, octapeptidyl removal by the mitochondrial intermediate peptidase (MIP) was ruled out, since no species of such an intermediate size were observed and no homology to known MIP substrates was found. The specificity of cleavage in the [Delta]77 region yet remains to be determined. The fact that UNG1[Delta]77 is present in human placental extracts (9), in the recombinant insect cells and apparently in intact HeLa mitochondria may indicate a protease of similar specificity. However, since UNG is normally not present in the insect cells, this protease is likely to have a more generalized function. This is also supported by the apparent susceptibility to general proteolytic cleavage in the [Delta]77 region as demonstrated by proteinase K cleavage. Two enzymatically active UDGs have also been demonstrated in rat liver mitochondria, one of which is apparently formed by proteolysis of the other (11). This may support the view that mammalian mitochondria contain two distinct functional UDGs. Interestingly, however, both the rat mitochondrial UDGs were inhibited by AP sites (11) and may reflect variant functions of the rat and human enzymes. The differential inhibition by AP sites and the identification of a specific protein-binding region in the N-terminal presequence of UNG1[Delta]29 may indicate that the 26 and 31 kDa UNG1 forms serve distinct functions. Both forms are, however, stimulated by HAP1, an AP endonuclease succeeding UNG proteins in the BER pathway. We have recently demonstrated that the rate-limiting step in catalysis by UNG1[Delta]84 is product release and that this may have a biological role in protecting cells from cytotoxic and mutagenic AP sites until further processed by HAP1 (25). It is thus tempting to speculate that the relative formation of 31 and 26 kDa forms of UNG1 may be regulated, perhaps to provide additional protection against the cytotoxic and mutagenic effects of unprocessed AP sites. Purified HAP1 was kindly provided by Dr Ian D. Hickson at the Institute of Molecular Medicine, University of Oxford. Thanks to Dr Knut Sletten at the University of Oslo Biotechnology Center who performed the N-terminal amino acid sequencing. This work was supported by The Norwegian Cancer Society, The Research Council of Norway and the Cancer Fund at the Regional Hospital in Trondheim.
RESULTS
Expression of human UNG in insect cells
Purification of UNG1 from insect cells
Human mitochondria contain two processed forms of UNG1, one of which is not formed by MPP
Catalytic properties of UNG1[Delta]29
UNG1[Delta]29 is not product inhibited by AP sites
UNG1[Delta]29 and UNG1[Delta]77 are stimulated by HAP1
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
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