Carcinogenesis, Vol. 22, No. 3, 387-393,
March 2001
© 2001 Oxford University Press
CANCER BIOLOGY |
Rates of base excision repair are not solely dependent on levels of initiating enzymes
DNA Repair Unit, Mutagenesis Laboratory, Istituto Nazionale Ricerca Cancro, Largo Rosanna Benzi n. 10, 16132 Genova, Italy,
1 Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555, USA and
2 Institute for Cancer Research and Molecular Biology, The Medical Department, Norwegian University of Science and Technology, 7489 Trondheim, Norway
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The oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG), the product of deamination of cytosine uracil (U), and the sites of base loss [abasic (AP) sites] are among the most frequent mutagenic lesions formed in the human genome under physiological conditions. In human cells, the enzymatic activities initiating DNA base excision repair (BER) of 8-oxoG, U and AP sites are the 8-oxoG DNA glycosylase (hOGG1), the U-DNA glycosylase (UNG) and the major hydrolytic AP endonuclease (APE/HAP1), respectively. In recent work, we observed that BER of the three lesions occurs in human cell extracts with different efficacy. In particular, 8-oxoG is repaired on average 4-fold less efficiently than U, which, in turn, is repaired 7-fold slower than the natural AP site. To discriminate whether the different rates of repair may be linked to different expression of the initiating enzymes, we have determined the amount of hOGG1, UNG and APE/HAP1 in normal human cell extracts by immunodetection techniques. Our results show that a single human fibroblast contains 123 000 ± 22 000 hOGG1 molecules, 178 000 ± 20 000 UNG molecules and 297 000 ± 50 000 APE/HAP1 molecules. These limited differences in enzyme expression levels cannot readily explain the different rates at which the three lesions are repaired in vitro. Addition to reaction mixtures of titrated amounts of purified hOGG1, UNG and APE/HAP1 variably stimulated the in vitro repair replication of 8-oxoG, U and the AP site respectively and the increase was not always proportional to the amount of added enzyme. We conclude that the rates of BER depend only in part on cellular levels of initiating enzymes.
Abbreviations: AP, abasic; APE/HAP1, human AP endonuclease; AU, absorbance units; BER, base excision repair; hOGG1, 8-oxoG DNA glycosylase; 8-oxoG, 8-oxo-7,8-dihydroguanine; U, uracil; UNG, U-DNA glycosylase; UNG
84, human recombinant UNG; UNG1, mitochondrial form of human UNG; UNG2, nuclear form of human UNG.
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Human 8-oxo-7,8-dihydroguanine (8-oxoG) DNA glycosylase (hOGG1), uracil (U) DNA glycosylase (UNG) and abasic (AP) endonuclease (APE/HAP1) are three important enzymes involved in base excision repair (BER), a major mechanism for repair of DNA damage of endogenous origin (reviewed in 1). The main function of hOGG1 is to remove the oxidized base 8-oxoG, an important premutagenic lesion that forms at a rate of ~1000 lesions per cell per day (2) and whose role in human mutagenesis and carcinogenesis has been repeatedly suggested (3,4).
UNG has evolved to counteract the mutagenic effects of U, several hundreds of which can be generated in DNA as a consequence of deamination of cytosine or misincorporation of dUTP during replication (1). hOGG1 is a bifunctional glycosylase, i.e. it is endowed with an AP lyase activity that incises in 3' the AP site resulting from the removal of 8-oxoG. In contrast, UNG is a monofunctional glycosylase, that can only remove U leaving a natural AP site as its end product. The latter must be incised subsequently by a major hydrolytic AP endonuclease termed APE, HAP1 or Ref-1 that copes with ~9000 AP sites per cell per day in the human genome (2). Therefore, hOGG1, UNG and APE/HAP1 are the starting enzymes that recognize and process three of the most frequent and miscoding lesions of endogenous origin. 8-oxoG, U and the natural AP sites are repaired with different efficiency in human cell extracts. The AP site is by far the most efficiently repaired lesion whereas 8-oxoG is the least (5). The efficiency of U repair is intermediate. Those differences in repair capacity might be linked to different expression of hOGG1, UNG and APE/HAP1 in human cells or to different kinetic features (or to both). The following experiments were performed to discriminate among the above possibilities.
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Cell culture and extracts
Normal human fibroblasts GM 5757 were obtained from the Human Genetic Mutant Cell Repository (Coriell Institute, Camden, NJ) and cultured as recommended.
Cell extracts were prepared by the method described by Biade et al. (6). Briefly, exponentially growing cells were harvested from 150 cm2 flasks, washed three times with phosphate-buffered saline (PBS) and resuspended in buffer I (10 mM Tris–HCl, pH 7.8 and 200 mM KCl), at a concentration of 5x107 cells/ml. An equal volume of buffer II (10 mM Tris–HCl, pH 7.8, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% Nonidet P-40, 2 mM dithio- threitol, 0.5 mM phenylmethyl sulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin) was then added and the cell suspension was stirred for 1 h at 4°C. The lysate was centrifuged at 16 000 g for 10 min and the supernatant dispensed into aliquots and stored in liquid nitrogen. The protein concentration ranged from 3.2 to 4.6 mg/ml.
Expression and purification of recombinant proteins
The hOGG1 cDNA (a gift from Dr Arthur Grollmann) was introduced into pET28a expression vector (Novagen, San Diego, CA). The hOGG1 open reading frame (NdeI/EcoRI) was inserted in-frame with an N-terminal 20 amino acid peptide containing a six-residue polyhistidine tag. Constructs encoding His-tagged hOGG1 proteins were expressed in Escherichia coli BL21 Codon Plus (Stratagene, La Jolla, CA) O/N at 16°C following induction with 0.1 mM (final concentration) IPTG at an OD600 of 0.3–0.4. Cells were harvested by centrifugation and disrupted on ice by sonication with a Braun Sonic U using maximum power. Total cell extracts were centrifuged for 15 s at 10 000 r.p.m. and His-tagged proteins in soluble fractions were applied to NTA–agarose (Qiagen, Santa Clarita, CA) and affinity purified. Eluted proteins were dialyzed against 50 mM NaCl, 20 mM Tris pH 7.4 and 10% glycerol and loaded onto HiTrapSP (Pharmacia, Uppsala, Sweden) for further purification. Protein was eluted at 300–350 mM NaCl and stored at –80°C. Its molecular mass was 42 kDa.
Human recombinant UNG (UNG84) (molecular mass: 27 kDa) lacking the first seven non-conserved N-terminal amino acids of the catalytic UNG domain was expressed in E.coli and purified as described previously (7).
Affinity purified human APE/HAP1 protein (molecular mass: 37 kDa) was provided by Novus Biologicals (Littleton, CO). It was prepared as described by Duguid et al. (8).
Polyclonal antibodies
A polyclonal antibody raised in rabbits against the whole hOGG1 protein was purchased from Alpha Diagnostic International (San Antonio, TX). A polyclonal antibody against UNG84 (termed PU101) was raised and purified as described by Slupphaug et al. (7). A polyclonal antibody raised in rabbits against affinity-purified APE/HAP1–GST fusion protein was purchased from Novus Biologicals.
Immunodetection of hOGG1 UNG and APE/HAP1
Twenty-five microliters of protein solution at the indicated concentrations and an equal volume of GM 5757 extract were loaded onto a discontinuous 5–12% SDS–PAGE minigel and electrophoresed at 90 V for 15 min followed by 180 V for 1 h. Proteins were electroblotted onto Hybond-C extra nitro- cellulose membrane (Amersham, Milano, Italy) in the presence of 25 mM Tris, 192 mM glycine, 20% methanol for 1 h at 100 V. Membranes were stained with Ponceau Red to check the blotting efficiency, washed with distilled water and blocked at 4°C overnight in PBS or Tris-buffered saline containing 0.1% Tween plus 5% dried skimmed milk. Membranes were incubated with polyclonals anti-hOGG1 (1:750 dilution) ), anti-UNG (1:2000 dilution) or anti-APE/HAP1 (1:1000 dilution) for 1 h at room temperature and subsequently with peroxidase-conjugated goat anti-rabbit IgG (whole molecule, Sigma, St Louis, MO) at a dilution of 1:2500 for 1 h at room temperature. Immune complexes were visualized by the enhanced chemiluminescence (ECL) system (Amersham).
Quantification of hOGG1 UNG and APE/HAP1
The intensity of bands was determined by scanning densitometry and expressed as absorbance units (AU)*mm. The molar concentrations of hOGG1, UNG and APE/HAP1 in extracts were determined using the regression equation calculated on the linear portion of the graph describing the absorbance of bands of purified proteins in the same experiment as a function of their molar concentration. The number of enzyme molecules per cell was calculated taking into account that 1µl extract was prepared from 25 000 cells.
Effect of purified hOGG1, UNG and APE/HAP1 on the efficiency of repair of 8-oxoG, U and AP sites
The in vitro repair of 8-oxoG, U and AP site was analyzed as described (5). Briefly, 300 ng of plasmid substrates containing a single 8-oxoG, U or AP site at a defined location (termed pGEM-8oxoG, pGEM U, pGEMX, respectively) or no damage (termed pGEM T) were incubated with 30 µg of GM 5757 extract protein for 3 h at 30°C in the presence of the indicated molar concentrations of hOGG1, UNG84 and APE/HAP1. [32P]dGTP was the labeling nucleotide when BER of a single 8-oxoG was under investigation whereas [32P]dTTP was the labeling nucleotide on pGEM U and pGEM X. After the repair reaction, the DNA reaction product was purified and treated with the restriction endonucleases XbaI–HincII. The repair incorporation in the resulting 8mer fragment is mostly derived from one nucleotide insertion BER (9). The amount of repair replication (expressed as Net CPM of [32P]dNMP incorporated) was quantified by densitometric scanning of autoradiographic bands. An hOGG1 enzyme with no polyhistidine tag was used in these experiments. The enzymatic activities of hOGG1, UNG84 and APE/HAP1 were determined as described (10,11,17).
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hOGG1
Based on sequence homology with yeast Ogg1, the cDNA of hOGG1 has been cloned independently in other studies (12–14). The hOGG1 gene is composed of eight exons, and several splicing isoforms have been identified (12,15). The different mRNAs resulting from differential splicing are expected to encode different hOGG1 polypeptides that each possesses a unique C-terminus (15). Indeed, enzyme purification yielded a major 8-oxoG glycosylase form of 38 kDa (16,17) together with other minor forms that are encoded by alternatively spliced hOGG1 mRNAs and have different intracellular localization (15).
Figure 1
shows a typical quantification experiment of hOGG1 by immunodetection. Twenty-five microliters of GM 5757 extract, containing 80 µg of protein, were loaded in lanes 1 and 2. A major band with an apparent molecular mass of 38 kDa was observed in the extracts together with a number of additional bands, which most likely correspond to alternatively spliced forms of hOGG1 (15). Pure hOGG1 (25 µl at concentrations of 0.5, 1, 5, 10, 50x10–9 M) was resolved in lanes 3–7. This protein shows a molecular mass of 41–42 kDa for the presence of a polyhistidine tag. Data were quantified by the plot relating the absorbance of bands of pure hOGG1 (lanes 3–7) to its molar concentration (Fig 1B). The equation describing the linear part of the graph is y = –0.17 + 0.19x. The r2 coefficient was 0.98. Using this equation, the concentration values of the major 38 kDa hOGG1 band in the two lanes of GM 5757 extract (lanes 1 and 2) were 3.38 and 3.16x10–9 M, respectively. It must be noted that although direct comparison of bands of extracts and the pure protein may suggest a higher concentration value for the 38 kDa form in extracts (compare the major band in lanes 1 and 2 with that in lane 5), this approach may be misleading due to minor deviations of experimental points obtained with the pure protein from the linear arrangement. When all bands were considered, the values of hOGG1 concentration in extracts increased to 8.09 and 8.44x10–9 M.
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A total of 14 experiments were performed (Table I
). The mean value of concentration ± standard error of the mean (SEM) was 1.91 ± 0.36x10–9 M for the major 38 kDa band and 5.10 ± 0.91x10–9 M considering all splicing forms. This corresponds to 46 000 ± 9000 and 123 000 ± 22 000 hOGG1 molecules per cell, respectively. The 38 kDa form represented therefore 46 000/123 000 = 37% of total hOGG1.
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UNG
The human UNG gene consists of seven exons and encodes both the nuclear (UNG2) and mitochondrial (UNG1) forms of the enzyme (18). The two forms are generated by alternative splicing and the use of two promoters (19). Both UNG2 and UNG1 can be detected in proliferating tissues, whereas UNG1 is the predominant form in tissues with low or no proliferation (20). Nuclear UNG2 differs from the mitochondrial form UNG1 in 44 amino acids of the N-terminal sequence that is not necessary for catalytic activity (19). UNG2 has a mass of 34.6 kDa. UNG proteins, however, migrate as slightly larger proteins in SDS–PAGE (18) and UNG2 migrates as a protein of ~38 kDa. This form is the major species in most tissues, representing ~70% of the total UNG activity (18). UNG1 is translated and rapidly imported into the mitochondria where it is cleaved to a species of 31 kDa that represents ~20% of the total UNG activity (21). Furthermore, both UNG2 and UNG1 can be cleaved by either mitochondrial or lysosomal proteases to a same species of 27 kDa.
The UNG concentration in GM 5757 extracts was quantified using as standard protein a recombinant UNG (UNG84) that closely resembles the mature form of the human enzyme (7). A representative quantification experiment is shown in Figure 2B. Twenty-five microliters of GM 5757 whole cell extracts were fractionated in lanes 1 and 2 and 25 µl of purified UNG84 protein at the indicated concentrations were resolved in lanes 3–6. Three bands were detected in extracts that corresponded to the 38 kDa UNG2 form (lanes 1 and 2, upper band), the 31 kDa UNG1 form (intermediate band) and the 27 kDa form derived from protease-cleavage (lower band).
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The relationship between absorbance of bands of UNG
84, as determined by scanning densitometry (lanes 3–6), and protein concentration was plotted (Figure 2B
). The equation describing the linear part is y = –0.452 + 0.635x (r2 = 0.97). Using this equation, the values of UNG2 protein concentration in the two lanes of GM 5757 extract (lanes 1 and 2) in this experiment were 2.97 and 2.75x10–9 M respectively. The concentrations of the 31 kDa (UNG1) form were 1.04 and 1.03x10–9 M, respectively. Those of the lower band form were 1.24 and 1.11x10–9 M, respectively. Total UNG concentration values were 5.25 and 4.89x10–9 M.
Fourteen experiments were performed (Table I). Mean ± SEM for total UNG from these 14 experiments was 7.39 ± 0.82x10–9 M corresponding to 178 000 ± 20 000 molecules per cell. Sixty-nine percent of UNG protein was nuclear (UNG2) whereas approximately half of the remaining 31% was mitochondrial (UNG1) and the other half was derived from proteolytic digestion.
APE/HAP1
The gene for APE/HAP1 has been cloned in different studies (22–25). It codes for a 37 kDa protein with strong sequence similarity to E.coli exonuclease III, with which it shares 30% sequence identity and 50% sequence similarity. Western analysis of HAP1 protein in human cell extracts detects a single protein of Mr = 37 000 (26).
An immunodetection experiment of APE/HAP1 in GM 5757 extracts is shown in Figure 3A. Similarly to the above experiments with hOGG1 and UNG, 25 µl (115 µg protein) of GM 5757 extract were fractionated in lanes 1 and 2 and 25 µl of APE/HAP1 solution at concentrations of 5, 10, 50 and 100x10–9 M were resolved in lanes 3–6, respectively. A single band of Mr = 37 000 was detected in extracts (lanes 1 and 2). The purified APE/HAP1 polypeptide produced by cleaving from a GST fusion protein migrated slightly faster than the extract protein. This could be due to a difference in the phosphorylation status of the two proteins [the extract APE/HAP1 can be phosphorylated (27) unlike the recombinant one], or simply to delayed migration of APE/HAP1 in extracts due to the large amount of proteins loaded in the wells. The plot describing the AU*mm of purified APE/HAP1 bands as a function of protein concentration is shown in Figure 3B. The equation describing the linear regression is: y = –0.389 + 0.091x. The r2 coefficient of this equation was 0.99. The values of APE/HAP1 concentration in GM 5757 extracts as determined by the intensity if bands in lanes 1 and 2 were 17.99 and 15.58x10–9 M, respectively.
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A total of 14 experiments were carried out (Table I
). The average concentration value ± SEM was 12.32 ± 2.07x 10–9 M that corresponds to 297 000 ± 50 000 APE/HAP1 molecules per cell.
Effect of purified hOGG1, UNG and APE/HAP1 on the in vitro repair of 8-oxoG, U and AP sites
A direct way to address the question of whether hOGG1, UNG and APE/HAP1 may be rate-limiting during BER of 8-oxoG, U and the AP site is to add titrated amounts of each recombinant protein to in vitro repair reactions and look at the extent to which repair efficiency is increased. One experiment of this kind is shown in Figure 4. Figure 4A shows a schematic representation of pGEM 8-oxoG, pGEM U, pGEMX and pGEM T (control) plasmid substrates with relevant restriction sites XbaI and HincII. Characterization of these substrates has been described previously (5,9). pGEM 8-oxoG (Figure 4B, lanes 1–3), pGEM U (lanes 4–6) and pGEMX (lanes 7–9) plasmid substrates were incubated with 30 µg protein of GM 5757 extract for 3 h in the presence of the indicated final concentrations of hOGG1 (lanes 2 and 3), UNG84 (lanes 5 and 6) and APE/HAP1 (lanes 8 and 9). The quantification of repair synthesis (Net CPM of [32P]dNMP incorporated, bottom of Figure 4B) shows that the repair replication stimulated by a single 8-oxoG (lane 1, black column) is less efficient (3.5-fold) than that stimulated by a single U (lane 4, hatched column) in agreement with a previous experiment (5). In turn, the latter is repaired less efficiently (4.5-fold) than a single AP site (lane 7, white column). Addition of purified active hOGG1 at 10x10–9 M final concentration (lane 2, black column) stimulated 7.6-fold the poor in vitro repair of 8-oxoG while a micromolar concentration (1000x10–9 M; lane 3, black column) produced a more limited increase (2.7-fold). Addition of purified UNG84 (10x10–9 and 1000x10–9 M; lanes 5 and 6, hatched columns) increased repair replication of U of 2.5- and 3.4-fold, respectively. Addition of 10x10–9 M APE/HAP1 (lane 8, white column) slightly improved (1.4-fold) the repair replication of a single AP site while an inhibitory effect (2.2-fold decrease) was observed with the micromolar concentration (lane 9, white column). No damage-independent incorporation was observed on pGEM T control substrates (lane 10).
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Recent work has shown that endogenous lesions to DNA are repaired with varying efficacy by human cell extracts. In particular, the oxidized base 8-oxoG poorly stimulates DNA repair replication in an in vitro BER assay (5,28) in comparison to U and the natural AP site. Repair incorporation stimulated by 8-oxoG is on average 4-fold lower than that stimulated by U, which in turn is repaired 7-fold less efficiently than a single AP site (ref. 5 and this study). BER of 8-oxoG and U is initiated in human cells by the two major specific glycosylases hOGG1 and UNG, whilst repair of natural AP sites is started by the major hydrolytic endonuclease APE/HAP1. In order to discriminate whether the different repair rates of 8-oxoG, U and the natural AP site could be linked to different expression levels of hOGG1, UNG and APE/HAP1, we have determined the number of molecules of these repair proteins per normal human fibroblast.
APE/HAP1 was the most abundant enzyme (297000 molecules per cell). This estimate was in agreement with the number of APE/HAP1 molecules (350 000 molecules per cell) in normal human fibroblasts obtained in another laboratory (29,30). The limited difference may be explained by the different quantification techniques as well as the different cell strains employed (29,30). A unique band was detected, in agreement with previous reports showing that no isoforms of APE/HAP1 exist in human cells (8,26).
UNG was expressed at a level of 178 000 molecules per cell. Most of them (69%) were represented by the 38 kDa UNG2 nuclear form in agreement with previous determinations (18). The ratio APE:UNG in terms of number of molecules per cell (1.7:1) was much lower than the ratio of in vitro repair replication efficiencies of human cell extracts on the AP site and U [7:1 (ref. 5 and this study)] thus indicating that the differences in repair replication efficiency depend on the level of initiating enzyme to only a limited extent.
hOGG1 was expressed at the lowest level. Six isoforms of this enzyme, probably generated via alternative splicing (15), could be recognized by an anti-hOGG1 polyclonal antibody raised against the whole protein, but a major band of Mr = 38 000 was predominant. This probably corresponds to the major form cloned by Radicella et al. (13) and purified from human nuclear extracts by Nishioka et al. (15). With respect to the other isoforms, one or more probably reside in mitochondria where BER of 8-oxoG has been documented (31). We estimated 46 000 ± 9000 38 kDa hOGG1 molecules per human fibroblast and 123 000 ± 22 000 molecules per cell when all isoforms were added together. The number of hOGG1 molecules per cell that we found in GM 5757 fibroblasts was 1.45- and 2.41-fold lower with respect to that of UNG and APE/HAP1, thus only partially explaining the reduced ability of human cell extracts to perform repair replication of 8-oxoG in comparison to U and the AP site [4- and 28-fold, respectively (ref. 5 and this study)].
Addition of titrated amounts of pure enzymes to reaction mixtures had different effects on repair of 8-oxoG, U or the natural AP site (Figure 4B).
Purified APE/HAP1 at nanomolar concentrations (lane 8) could only slightly (1.4-fold) stimulate the repair of a single AP site while an inhibiting effect was observed at micromolar concentrations (lane 9). This suggests that the endogenous supply of APE/HAP1 may be close to optimum levels in extracts, in accordance with the elevated levels observed (297 000 molecules per cell), and that high enzyme concentrations may in fact interfere with the repair reaction.
Addition of pure UNG84 at both nanomolar and micromolar concentrations (lanes 5 and 6) consistently stimulated the repair replication of pGEM U 2–3-fold. This indicates that although high (178 000 molecules per cell), extract levels of UNG may still limit BER of U in vitro.
Addition of purified hOGG1 at nanomolar concentrations could significantly elevate the level of repair incorporation at the single 8-oxoG (lane 2) while micromolar concentrations were less effective (lane 3). Hence, the efficiency of BER of 8-oxoG in vitro is determined in part by the level of hOGG1 molecules. Yet, it is unlikely that this is the only factor involved and the slow kinetic properties of hOGG1 may also play a role in the inefficient repair of 8-oxoG. Jaruga and Dizdaroglu (32) have determined the in vivo repair of 11 modified bases produced upon hydrogen peroxide treatment of human cells. In general, repair of purine lesions was slower than that of pyrimidine lesions and 8-oxoG was the second most persistent adduct, thus indicating that hOGG1 may have poor glycolytic capacity. Poor kinetic properties of hOGG1 have been described recently by Asagoshi et al. (33). In this study, the repair activities of hOGG1 and its E.coli counterpart, formamidopyrimidine DNA glycosylase (Fpg), were compared using oligonucleotides containing a single 8-oxoG as substrates. The kcat/Km values of hOGG1 were ~80-fold lower than those of Fpg. Lower substrate affinity and slower hydrolysis of the Schiff base intermediate by hOGG1 in comparison to Fpg were demonstrated. Hence, the low efficiency of repair of 8-oxoG in human cell extracts may be determined, at least in part, by the sluggish kinetic properties of hOGG1. In addition to inherent inefficacy, low hOGG1 activity may also be due to the presence of an 8-oxoG-specific DNA binding protein (17). It cannot be ruled out that the low repair replication of 8-oxoG in human cell extracts may in part be due to specific inactivation of the hOGG1 protein at the moment of extract preparation. Yet, this is unlikely. First, poor repair of 8-oxoG can also be observed in vivo, as mentioned above (32). Second, hOGG1 showed no particular lability during purification procedures (14,17) and poor 8-oxoG repair replication could be observed with extracts gently prepared by the procedure of Manley et al. (34) that are competent for complex DNA transactions such as nucleotide excision repair and transcription (28; E.Cappelli and G.Frosina, unpublished results). Third, a predominant form with the expected Mr of 38 000 was detected in our extracts, thus indicating that no extensive degradation of the protein occurred.
We can conclude that the different repair rates of 8-oxoG, U and AP site are determined only in part by the cellular levels of hOGG1, UNG and APE/HAP1.
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Notes |
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3 To whom correspondence should be addressed Email: gfrosina{at}hp380.ist.unige.it
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Acknowledgments |
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We thank Ottavio Rossi for preparation of pGEM plasmid substrates. This work was partially supported by the Italian Association for Cancer Research (AIRC), Telethon Italy, the National Research Council [grant no. 99.02487.CT04 (*)] and the Italian Ministry of Health. M.B. was the recipient of an Italian Foundation for Cancer Research fellowship.
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