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Originally published In Press as doi:10.1074/jbc.M411864200 on March 3, 2005

J. Biol. Chem., Vol. 280, Issue 18, 18469-18475, May 6, 2005
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DNA Polymerase {lambda} Mediates a Back-up Base Excision Repair Activity in Extracts of Mouse Embryonic Fibroblasts*

Elena K. Braithwaite, Rajendra Prasad, David D. Shock, Esther W. Hou, William A. Beard, and Samuel H. Wilson{ddagger}

From the Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, October 19, 2004 , and in revised form, February 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian DNA polymerase (pol) {lambda} is a member of the X-family of DNA polymerases and has striking enzymatic and structural similarities to mammalian DNA pol {beta}. Because pol {beta} provides two important enzymatic activities for base excision repair (BER), we examined whether pol {lambda} might also contribute to BER. We used extracts from mouse embryonic fibroblasts representing wild-type and null genotypes for pol {beta} and pol {lambda}. In combination with neutralizing antibodies against pol {beta} and pol {lambda}, our results show a BER deficiency in the pol {lambda} -/- cell extract compared with extract from isogenic wild-type cells. In addition, the pol {lambda} antibody strongly reduced in vitro BER in the pol {beta} -/- cell extract. These data indicate that pol {lambda} is able to contribute to BER in mouse fibroblast cell extract.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells are constantly exposed to environmental stress agents, endogenous reactive oxygen species and alkylating molecules, and other reactive metabolites that are capable of modifying DNA. Cells have several mechanisms by which they protect themselves from the detrimental effects of genotoxic compounds. Base excision repair (BER)1 is the predominant DNA repair system in mammalian cells for eliminating discrete DNA base lesions (1). Single-nucleotide BER, one subpathway of BER, results in replacement of only the modified nucleotide and is initiated by a lesion-specific DNA glycosylase. Monofunctional DNA glycosylases hydrolyze the glycosidic bond between the sugar and base of the damaged nucleotide, whereas bifunctional DNA glycosylases excise the damaged base and cleave the phosphodiester backbone 3' to the resulting abasic site. In the former case, the resulting apurinic/apyrimidinic site is cleaved by apurinic/apyrimidinic endonuclease (APE), producing a single-strand DNA break. DNA polymerase gap-filling DNA synthesis and 5'-deoxyribose phosphate (dRP) removal generates the substrate for the final BER step, DNA ligation. In higher organisms, DNA polymerase (pol) {beta} is thought to be the primary DNA polymerase recruited for BER of lesions generated by monofunctional alkylating agents and reactive oxygen species (2-7). Pol {beta} contributes both gap-filling DNA synthesis and 5'-dRP lyase activities to the overall BER reaction. However, the contribution of other DNA polymerases to BER has been well documented both in vitro (4, 8) and in vivo (9). Another candidate BER DNA polymerase, DNA pol {lambda}, has been identified that shares many biochemical features with pol {beta} and Saccharomyces cerevisiae DNA pol IV (10-13). Like pol {beta}, pol {lambda} is a member of the X-family of DNA polymerases and shares substantial amino acid sequence identity with pol {beta} (10, 11, 14). Sequence and structural analyses reveal that pol {lambda} contains both of the pol {beta} functional domains, the polymerase domain with functionally distinct subdomains and the dRP lyase domain. Many amino acid residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis that have been identified for pol {beta} are conserved in pol {lambda} (10, 15, 16). In fact, pol {lambda} shares two key BER characteristics with pol {beta}, gap-filling DNA synthesis activity and dRP lyase activity (16, 17). These activities allow pol {lambda} to substitute for pol {beta} in a single-nucleotide BER system reconstituted with purified human proteins (17). As with pol {beta}, pol {lambda} is a distributive enzyme on open (i.e. not gapped) template primer substrates; however, it is processive on substrates containing a small gap with a 5'-phosphate group (16).

Pol {lambda} contains a relatively long N-terminal region that has no counterpart in pol {beta}. This region is similar to the N-terminal region of S. cerevisiae DNA pol IV and contains a BRCA1 C-terminal domain and serine-threonine-proline-rich domain (10, 14). Although the functions of this N-terminal region are not yet clear, it could promote enzyme localization through protein-protein interactions and could also be a target for post-translational modifications (10). The idea that the BRCA1 C-terminal domain may facilitate protein-protein interactions is supported by a previous study showing that the BRCA1 C-terminal domain is required for efficient gap-filling activity by pol {lambda} and S. cerevisiae DNA pol IV during non-homologous end-joining in vitro (18-20). Other biochemical properties of the enzyme support a role for pol {lambda} in non-homologous end-joining (21, 22). Despite the extensive analyses of pol {lambda} so far, its possible cellular functions in BER are not yet established. Although in vitro studies have clearly shown that the purified pol {lambda} enzyme is capable of supporting BER reactions, it remains unclear whether the enzyme is expressed in sufficient amounts to support BER in the cell or whether the enzyme is modified in a manner that would prevent its involvement in the BER process. In an effort to further clarify the role of this enzyme in BER, we performed a series of in vitro BER assays with extracts from genetically modified mouse cells lacking functional pol {lambda} alleles.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Synthetic oligodeoxyribonucleotides were from Oligos Etc, Inc. [{alpha}-32P]dCTP (3000 Ci/mmol) was from GE Healthcare. [{gamma}-32P]ATP (7000 Ci/mmol) was from MP Biomedicals. Polynucleotide kinase was from New England Biolabs. Human APE (23), pol {beta} (24, 25), human uracil-DNA glycosylase (UDG) (26) and DNA ligase I (27) were purified as described previously. Monoclonal (18 S) and polyclonal antibodies against pol {beta} were developed as reported previously (6), whereas antibodies specific for full-length human pol {lambda} were raised for this study by immunization of rabbits as described previously (28). The mouse and rabbit IgG secondary antibodies were goat anti-mouse IgG horseradish peroxidase conjugate and goat anti-rabbit IgG horseradish peroxidase conjugate, respectively, and both were from Bio-Rad. Pol {lambda}-neutralizing antibodies were a generous gift from Drs. Miguel García-Díaz and Luis Blanco (Centro de Biología Molecular Severo Ochoa). Bacterial expression cloning vector, pET28a (+), and His-Bind purification kit were from Novagen. Bacterial competent cells (BL21 codon plus(DE3) RP and XL1-Blue) and Pfu Turbo DNA polymerase were from Stratagene. Dimethyl sulfoxide (Me2SO) was from Sigma-Aldrich and Lipofectamine reagent was from Invitrogen.



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  		FIG. 1.
Characterization of pol {lambda} and pol {beta} wild-type and null MEF cell extracts. The conditions and product analyses were as described under "Experimental Procedures." A, photographs of enhanced chemiluminescence-stained immunoblots are shown. MEF cell extracts were separated by SDS-PAGE, transimmunoblotted onto a nitrocellulose membrane, and these were reacted with antibody specific to pol {lambda}, pol {beta}, or actin, as indicated. Lane 5 contains the respective purified enzyme as a positive control. B and C, in vitro BER time course showing the capacity of wild-type or pol {beta}-null cell extracts (B) or wild-type and pol {lambda}-null cell extracts (C), respectively. The BER reaction mixtures were incubated for 10 (lanes 1 and 5), 30 (lanes 2 and 6), 60 (lanes 3 and 7) and 120 min (lanes 4 and 8) and product analyses were performed as described under "Experimental Procedures." Photographs of autoradiograms, after denaturing PAGE illustrating the incorporation of [32P]dCMP into DNA, are shown. The positions of the ligated BER product (35-mer) and unligated one-nucleotide (1-nt) insertion product (15-mer) are indicated. D, gel migration behavior of oligonucleotide standards corresponding in sequence to the 15-mer product (lane 1), 35-mer product (lane 2), and DNase I controlled digestions: short (5 min, lane 3) and long (10 min, lane 4). Lengths of the digestion products are shown.

 
Cell Lines—Pol {beta} +/+ cells used were a clone of the wild-type mouse embryonic fibroblast (MEF) cell line M{beta} 16tsA. Pol {beta} -/- cells were a clone of the isogenic pol {beta}-null line M{beta} 19tsA (2). Pol {lambda} +/+ and -/- primary MEFs were a generous gift from Drs. Barbara Bertocci and Claude-Agnès Reynaud (Institut National de la Santé et de la Recherche Médicale, Paris, France) and were generated from mice after a targeted pol {lambda} gene deletion (29). Pol {lambda} +/+ and -/- primary MEFs were grown at 37 °C in a 10% CO2 incubator in Dulbecco's modified Eagle's medium containing GlutaMAX-1 and 10% fetal bovine serum.

Transformation of Primary Pol {lambda} +/+ and Pol {lambda} -/- Cells—To establish cell lines, subconfluent cultures of primary cells were transfected with the pSVAgt plasmid, which expresses simian virus 40 T antigen. Cells were incubated with Lipofectamine solution containing serum-free growth medium and pSVAgt plasmid DNA for 17 h at 37 °C. The Lipofectamine solution was then replaced with growth medium, and the cells were cultured at 37 °C for an additional 48 h. Cells were repeatedly split at a ratio of 1:20 after reaching confluence until characteristics of transformed cells were achieved (i.e. fast growing, change in cell morphology, lack of contact inhibition). Finally, cells were single-cell cloned by plating at a density of 0.1 to 1 cell per well in a 96-well dish. Four such clones were selected from each cell line.

Cell Extract Preparation—Extracts were prepared as described previously (30). In brief, cells were washed twice with phosphate-buffered saline at room temperature, detached by scraping, pelleted by centrifugation, and resuspended in Buffer I (10 mM Tris-HCl, pH 7.8, 200 mM KCl, and protease inhibitor mixture (Roche Molecular Biochemicals)). 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, and 2 mM dithiothreitol) was added. The suspension was rotated for 1 h at 4 °C, and the resulting extract was clarified by centrifugation at 20,800 x g (average) for use in Western blotting and in vitro BER assays. The protein concentration of the extract was determined by Bio-Rad protein assay analysis using bovine serum albumin as a standard.

Western Blotting—Western blotting analysis was performed as described previously (31). Cell extract (30 µg) was separated by Nu-PAGE 4-12% Bis-Tris mini-gel (Invitrogen) and transferred electrophoretically to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% (v/v) Tween 20 and then incubated for 2 h with primary antibodies against pol {beta}, pol {lambda}, or actin. After washing, the membranes were treated with horseradish peroxidase-conjugated secondary antibodies. Horseradish peroxidase activity was measured by an enhanced chemiluminescence system (GE Healthcare).

In Vitro BER Assay with Cell Extract—Unless specified otherwise, 10 µg of cell extract were analyzed using a single-nucleotide BER assay in a final volume of 10 µl (32). A 35-base-pair oligonucleotide duplex (250 nM) containing uracil at position 15 was preincubated with cell extract for 5 min at 37 °C in a BER reaction buffer containing 50 mM HEPES, pH 7.5, 0.5 mM EDTA, 2 mM dithiothreitol, 20 mM KCl, 4 mM ATP, 5 mM phosphocreatine, 100 µg/ml phosphocreatine kinase, and 0.5 mM NAD. The repair reaction was then initiated by adjusting the final reaction mixture to 10 mM MgCl2, and 2.2 µM [{alpha}-32P]dCTP (specific activity, 1 x 106 dpm/pmol). In addition, some reactions were supplemented with 10% (v/v) Me2SO. The reaction mixture was incubated at 37 °C for 10 min, and BER products were measured as described previously (32). In vitro BER time course reactions were performed in a similar manner. 15 µg of cell extract were analyzed using a single-nucleotide BER assay in a final volume of 15 µl. A 35-base-pair oligonucleotide duplex (250 nM) containing uracil at position 15 was incubated with cell extract in the BER reaction buffer described above supplemented with 10 mM MgCl2 and 2.2 µM [{alpha}-32P]dCTP (specific activity, 1 x 106 dpm/pmol). The reaction mixture was incubated at 37 °C, and 3-µl aliquots of the reaction mixture were removed for analysis at the indicated times. BER products were measured as described previously (32). A reconstituted BER reaction was also performed under similar reaction conditions as above using the recombinant human enzymes including 10 nM UDG, 10 nM APE, 200 nM DNA ligase I, and 10 nM pol {beta}, or 100 nM pol {lambda}. BER products were measured as described previously (33).



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  		FIG. 2.
Use of pol {beta}-neutralizing antibody to eliminate pol {beta}-dependent repair in the MEF extract. The conditions and product analyses were as described under "Experimental Procedures." A, extract (10 µg) from pol {lambda} +/+ cells was incubated after addition of buffer (lane 1), preimmune serum (lane 2), or immune serum against pol {beta} (lane 3) for 30 min on ice and an additional 10 min at 25 °C. After preincubation, uracil-containing 35-base-pair duplex DNA was added, and repair reactions were performed as described under "Experimental Procedures." The reaction products were analyzed as in Fig. 1. B, increasing concentrations of pol {beta}-neutralizing antibody was preincubated with pol {beta} +/+ extract, and in vitro BER reactions were performed as described previously. Reaction products were separated by denaturing PAGE, and the gels were scanned by a PhosphorImager. Ligated BER products were quantified by using ImageQuant software. Products (PhosphorImager units) were plotted as a function of antibody concentration. C, results of reconstituted BER with purified enzymes, illustrating that the neutralizing antibody against pol {beta} almost completely neutralized repair activity of pol {beta} but did not influence the repair activity of pol {lambda}. In vitro BER reaction was reconstituted with 10 nM UDG, 10 nM APE, 200 nM DNA ligase I, and 10 nM pol {beta} (lanes 1 and 2) or 50 nM pol {lambda} (lanes 3 and 4). The reaction mixtures were preincubated with preimmune serum (lanes 1 and 3) or with immune serum against pol {beta} (lanes 2 and 4), as above. BER reactions and product analyses were performed as described above in Fig. 1. Photographs of autoradiograms are shown illustrating the incorporation of [32P]dCMP into DNA. The positions of the ligated BER product (35-mer) and unligated one-nucleotide (1-nt) insertion product (15-mer) are indicated.

 
Deoxyribonuclease I (DNase I) Treatment—The standard DNase I reaction was assembled on ice in a reaction (20 µl) that contained 50 nM 32P-labeled 35-base pair duplex DNA (BER substrate), 40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, and 2 µl of DNase I (1 unit diluted 1:100). The reaction mixture was incubated at 30 °C, and 10-µl aliquots were withdrawn at 5 min intervals. The reaction was stopped by addition of gel loading dye (10 µl), and the reaction products were separated as described previously (33). 32P-labeled 35-mer and 15-mer oligonucleotides were used as markers.

Construction of Expression Plasmid for Human Pol {lambda}—A full-length human pol {lambda} cDNA clone was purchased from Invitrogen and amplified by PCR using primer 1 (5'-CCGCGCGGCAGCCATATGGATCCCAGGGGTATCTTGAAGGCATTTCC-3') and primer 2 (5'-GCGGCCGCAAGCTTCTATCA1731CCAGTCCCGCTCAGCAGGTTCTCGGTAG-3') that introduced an NdeI restriction site (underline) with an initiation ATG codon (italics) and a HindIII restriction site (underline) with a double termination TGATAG codon (italics), respectively. The first and last nucleotides of the pol {lambda} sequence are numbered 1 and 1731, respectively. The PCR product was digested with the indicated restriction enzymes (NdeI and HindIII) and cloned into a pET28a (+) expression vector (Novagen). The resulting plasmid, pET-pol {lambda}, contained the full-length open reading frame for human pol {lambda} with a His6 tag at the N terminus. Plasmid pET-pol {lambda} was transformed into the XL1-Blue strain of Escherichia coli, DNA was extracted from five independent clones, and the sequence of pol {lambda} was confirmed by DNA sequencing.

Overexpression and Purification of Recombinant Pol {lambda} from pET-pol {lambda}—Escherichia coli strain BL21 codon plus (DE3) RP, transformed with pET-pol {lambda} was inoculated in 1.8 liters of Luria broth that contained 35 µg/ml kanamycin (MP Biomedicals) and 1% glucose to suppress target protein expression. Cells were grown overnight to an A595 nm of 2.0. Thereafter, the cells were collected by centrifugation and resuspended in fresh Luria broth containing kanamycin to an A595 nm of 1.0 and allowed to grow for 2 h at 18 °C. Isopropyl-{beta}-D-thiogalactopyranoside was added to a final concentration of 0.25 mM, and incubation was continued for 20 h at 18 °C. Cells were then harvested by centrifugation and resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM {beta}-mercaptoethanol, and protease inhibitors, including 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM benzamidine, 1 µg/ml pepstatin A, and 1 µg/ml leupeptin) containing 500 mM NaCl. The cell suspension was sonicated and clarified by centrifugation at 186,000 x g (average) for 30 min at 4 °C. The cell lysate was diluted with Buffer A to adjust the NaCl concentration to 100 mM and passed through a Q-Sepharose (GE Healthcare) column (6 x 14 cm) that was pre-equilibrated with Buffer A containing 100 mM NaCl. The flow-through fraction was collected and imidazole and NaCl were added to adjust the final concentrations to 5 and 500 mM, respectively. This fraction was then applied to a 5-ml column of nickel immobilized on His-Bind resin that was pre-equilibrated with Buffer B (Buffer A with 500 mM NaCl and 5 mM imidazole). The column was washed extensively in sequence with 12 column volumes each of Buffer B, Buffer B without imidazole but with 1 M NaCl, and Buffer A containing 100 mM NaCl and 5 mM imidazole. Proteins bound to the column were eluted with a linear 5-500 mM imidazole gradient in Buffer A containing 100 mM NaCl. Column fractions (3 ml) were collected, and {beta}-mercaptoethanol and EDTA were immediately added to each fraction to final concentrations of 7 and 0.5 mM, respectively. An aliquot (10 µl) of alternate fractions was analyzed using a Nu-PAGE 4-12% Bis-Tris mini-gel system and MOPS buffer. Fractions containing pol {lambda} (~67-kDa protein) were pooled and diluted 3-fold with Buffer A containing 100 mM NaCl to adjust the imidazole concentration to ~100 mM. The pooled fraction was applied to a RESOURCE S (6 ml) column, washed with Buffer C (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 7 mM {beta}-mercaptoethanol, 0.5 mM EDTA, and protease inhibitors) and eluted with an NaCl gradient (100 mM to 1 M) in Buffer C. Fractions were analyzed as described above, and those containing pol {lambda} were pooled. The pooled fraction was diluted with 50 mM Tris-HCl, pH 7.5, to adjust the NaCl concentration to 100 mM, and was applied to a single-stranded DNA-cellulose column (2.5 x 20 cm). After washing the column with Buffer C, the bound proteins were eluted using a linear 0.1-1 M NaCl gradient in Buffer C. Fractions containing pol {lambda} were pooled and concentrated, using Amicon Ultra-15 centrifugal filter devices (Millipore), to a desired concentration. During the concentration step, the protein sample was exchanged into storage buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM {beta}-mercaptoethanol, 0.5 mM EDTA, and 20% glycerol), frozen in dry ice, and stored at -80 °C. Protein concentration was determined by Bio-Rad assay.



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  		FIG. 3.
Use of pol {beta}-neutralizing antibody to illustrate pol {lambda}-dependent back-up BER in wild-type MEF extract. A, a neutralizing antibody against pol {beta} and pol {lambda} wild-type and null cell extract were used to demonstrate pol {lambda}-dependent BER activity in wild-type cell extract. Cell extracts from pol {lambda} +/+ (lanes 1-8) or pol {lambda} -/- (lanes 9-16) cells were incubated with preimmune serum (lanes 1-4 and 9-12) or with a neutralizing antibody against pol {beta} (lanes 5-8 and lanes 13-16) for 30 min on ice and then an additional 10 min at 25 °C. After the preincubation, uracil-containing 35-base-pair duplex DNA was added, and repair reactions were performed as described in Fig. 1. Aliquots of the reaction mixture were stopped at 10 (lanes 1, 5, 9, and 13), 30 (lanes 2, 6, 10, and 14), 60 (lanes 3, 7, 11 and 15), and 120 min (lanes 4, 8, 12, and 16). Reaction products were analyzed as described under "Experimental Procedures." The position of the ligated BER product (35-mer) is illustrated. B, ligated 35-mer BER products in lanes 5-8 and 13-16 were scanned by a PhosphorImager and quantified using ImageQuant software. Products (PhoshorImager units) were plotted against time.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro BER Assay with Uracil-containing Oligonucleotide Duplex DNA Substrate—Because cell extracts support a variety of biochemical activities, they can provide a window into the events within a cell and represent a more biologically relevant model for studying the biochemical activity of a given protein. Post-translational modification, protein expression, and the formation of protein complexes are all biochemical processes that have the potential to be analyzed in a cell extract. Therefore, in an effort to further examine pol {lambda} in the context of these processes, a series of MEF cell extract-mediated BER reactions were performed with uracil-containing oligonucleotides. First, the cell lines used in this study were characterized by Western blotting to examine expression levels of pol {beta} and {lambda}. As shown in Fig. 1A, pol {lambda} was absent in the pol {lambda} -/- cell line, as expected, and the level of pol {lambda} was equivalent in the pol {beta} +/+ and -/- cell lines. In addition, the level of pol {beta} was almost equivalent in the pol {lambda} +/+ and -/- cell lines. A 35-base pair oligonucleotide duplex containing uracil at position 15 was incubated with extracts prepared from these MEF cell lines. Incorporation of [32P]dCMP into the oligonucleotide substrate by the extract was used to monitor repair synthesis. At the indicated times, each reaction was stopped and analyzed as described under "Experimental Procedures." As expected from previous work (2), the pol {beta} -/- cell extract showed a strong BER deficiency in this assay, compared with extract from the wild-type cell line (Fig. 1B). However, pol {beta} -/- cell extract still retained significant repair activity, and this was clearly observed with longer incubation periods (Fig. 1B, lanes 6-8) suggesting the presence of a pol {beta}-independent BER back-up pathway. Extract from pol {lambda} -/- cells, on the other hand, showed no significant repair deficiency, compared with the wild-type cell line (Fig. 1C).

Conditions for Study of Pol {beta}-independent Back-up BER in MEFs—Because pol {beta} is such an active enzyme in the BER process, it is difficult to analyze the contribution of back-up BER polymerases in its presence. In an effort to overcome this obstacle, an antibody against pol {beta} was used to neutralize its activity and, therefore, analyze a possible role of pol {lambda} in BER. This pol {beta}-neutralizing antibody was characterized for its effectiveness and specificity. First, the antibody was tested for its ability to neutralize pol {beta} activity in a wild-type MEF extract. Whereas preimmune serum had little or no effect on BER activity (Fig. 2A), the pol {beta} antibody decreased DNA repair synthesis to a near-background level, similar to the results observed with pol {beta} -/- cell extract (compare Fig. 1B, lane 5, and Fig. 2A, lane 3). Note that these reactions were incubated for 10 min, and therefore the signal from back-up repair activity was negligible (Fig. 2A, lane 3). The concentration of antibody used in the reaction mixture (0.5 µg/µl) gave near-maximal inhibition of pol {beta} activity in a wild-type cell extract (Fig. 2B). Because both pols {beta} and {lambda} belong to the X-family and share many structural and biochemical properties, we wanted to ensure that the antibody inhibition was specific to pol {beta} and did not influence pol {lambda} activity. An in vitro BER system with purified enzymes including UDG, APE, pol {beta}, or pol {lambda} and DNA ligase I was used in this assessment (33). In reactions with pol {beta}, the antibody against pol {beta} strongly reduced both unligated (15-mer) and ligated (35-mer) BER products; however, in reactions with pol {lambda}, this antibody had little effect on DNA repair synthesis (Fig. 2C). These observations indicate that the neutralizing antibody against pol {beta} did not alter the activity of pol {lambda}.



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  		FIG. 4.
Use of Me2SO to stimulate pol {beta}-independent BER activity in pol {beta} -/- MEF extracts. The conditions and product analyses were as described under "Experimental Procedures." A, in vitro BER reactions were performed with increasing concentrations (5, 10, and 20 µg) of cell extract. Pol {beta} -/- cell extracts were preincubated with uracil-containing 35-base-pair duplex DNA. After preincubation, BER reactions were initiated by addition of MgCl2 and [{alpha}-32P]dCTP in either the absence (lanes 1-3) or the presence (lanes 4-6) of 10% (v/v) Me2SO. The repair reaction was allowed to proceed for 10 min at 37 °C. Reaction products were separated by 15% denaturing PAGE as described in Fig. 1. The position of the ligated product (35-mer) is indicated. B, after preincubation at the indicated DNA concentrations with 10 µg of pol {beta}-null cell extract, the repair reactions and product analyses were performed in the absence ({circ}) or in the presence () of Me2SO, as described in A. The gels were scanned by PhosphorImager, and the ligated BER products were quantified using ImageQuant software. Products (arbitrary PhosphorImager units) were plotted as a function of DNA concentration. C, in vitro BER time course reactions were performed in the absence (lanes 1-3) or presence (lanes 4-6) of 10% Me2SO for 10 (lanes 1 and 4), 60 (lanes 2 and 5), or 120 min (lanes 3 and 6). D, the reaction mixtures contained 10 nM UDG, 10 nM APE, 200 nM DNA ligase I, 10 nM pol {beta} or 100 nM pol {lambda}, as indicated, and 250 nM uracil-containing DNA. In vitro BER reactions were incubated in the presence (+) or absence (-) of 10% (v/v) Me2SO, as indicated. The position of the ligated product is shown.

 
Characterization of Pol {lambda}-mediated BER in the Absence of Pol {beta} Activity—Using the pol {beta} antibody, we characterized the role of pol {lambda} in MEF extract-mediated BER. Pol {lambda} -/- and isogenic wild-type MEF cell extracts were prepared and used in an in vitro BER assay for time course experiments. Cell extracts were preincubated with either preimmune serum or the antibody against pol {beta} (Fig. 3A). First, when the wild-type cell extract was preincubated with pol {beta} antibody, the level of BER decreased, but a substantial level of BER persisted that was measured at longer incubation periods (Fig. 3A, lanes 1-8). When a similar experiment was performed with pol {lambda} -/- cell extract, the level of total BER DNA synthesis, ligated and unligated BER products, was 2-fold lower than that observed with the pol {lambda} +/+ cell extract (Fig. 3A, compare lanes 5-8 with lanes 13-16). Therefore, when both pol {beta} and pol {lambda} activities were eliminated, by antibody and genetic manipulation, respectively, BER activity was lower than in the presence of either of these enzymes (Fig. 3A, compare lanes 5-8 and 13-16). It is interesting that in the absence of pol {beta} and {lambda}, these extracts were able to perform BER activity, albeit at a reduced level (Fig. 3B).

Improved Conditions for Study of Pol {beta}-independent in Vitro BER—Although a BER defect as a function of pol {lambda} was observed with the aid of the pol {beta}-neutralizing antibody, more optimal conditions for examining pol {beta}-independent BER were discovered during initial phases of this project. We first examined the effects of a series of DNA polymerase inhibitors in our in vitro BER assay to reveal an inhibition profile consistent with pol {lambda} (data not shown). During these preliminary studies, we observed that the solvent Me2SO stimulated pol {beta}-independent repair. As seen in Fig. 4A, an increase in BER activity in the pol {beta} -/- cell extract was observed when Me2SO was included in the assay mixtures that contained increasing amounts of cell extracts (compare lanes 1-3 with lanes 4-6). Without Me2SO, DNA repair remained at a background level at all concentrations of extract tested using a 10-min incubation period. This observation agrees with a previous study describing a stimulation of mammalian cell extract-mediated DNA repair synthesis by Me2SO (7). In addition, others have used Me2SO to stimulate DNA polymerase activity, presumably through a dielectric constant effect (34, 35). To further investigate the effect of Me2SO on pol {beta}-independent BER by the cell extract, we analyzed BER stimulation as a function of increasing DNA concentration in the reaction mixture (Fig. 4B). The rate of repair was dependent on the concentration of DNA substrate in the absence and presence of Me2SO, as expected (Fig. 4B); however, the maximal activity (i.e. plateau phase of the curves) was greater in the presence of Me2SO. The above results suggest that residual BER activity in the pol {beta} -/- extract, presumably pol {lambda}-mediated, was stimulated by Me2SO. The idea that Me2SO can stimulate pol {lambda}-dependent repair is supported by the observation that Me2SO did not have a significant impact on the level of BER activity in pol {lambda} -/- cell extracts (Fig. 4C). In addition, using a reconstituted BER system with purified proteins, Me2SO slightly stimulated the reaction containing purified pol {lambda}, whereas the system assembled with pol {beta} was inhibited (Fig. 4D).



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  		FIG. 5.
Use of Me2SO contribution and neutralizing antibodies to study the of pol {lambda} to in vitro BER in pol {beta} -/- cell extracts. A, the reaction mixtures contained 10 nM UDG, 10 nM APE, 200 nM DNA ligase I, 100 nM pol {lambda}, and 250 nM uracil-containing DNA. These purified proteins were preincubated with buffer alone (lanes 1-4), preimmune serum (lanes 5-8), or a neutralizing antibody against pol {lambda} and subsequently used in BER reactions containing 10% (v/v) Me2SO as described previously. The position of the ligated product is shown. B, cell extracts from pol {beta} -/- cells were incubated with buffer (lanes 1-4), preimmune serum (lanes 5-8), or with a neutralizing antibody against pol {lambda} (lanes 9-12) for 30 min on ice and then an additional 10 min at 25 °C. After the preincubation, uracil-containing 35-base-pair duplex DNA was added, and repair reactions were performed as described in Fig. 1. Aliquots of the reaction mixture were stopped at 10 (lanes 1, 5, and 9), 30 (lanes 2, 6, and 10), 60 (lanes 3, 7, and 11), and 120 min (lanes 4, 8, and 12). Reaction products were analyzed as described under "Experimental Procedures." The position of the ligated BER product (35-mer) is illustrated.

 
Further Characterization of Extract-mediated BER with Antibodies—With Me2SO to stimulate pol {beta}-independent repair, neutralizing antibodies against both pol {lambda} and pol {beta} were used to examine a role of pol {lambda} in extract-mediated in vitro BER. First, a pol {lambda}-neutralizing antibody was characterized using a reconstituted BER system to determine whether the antibody effectively inhibited pol {lambda}-dependent BER activity over the 2-h period used in our studies. As shown in Fig. 5A, preimmune serum had no significant effect on the BER activity of pol {lambda} (compare lanes 1-4 and 5-8). However, the pol {lambda}-neutralizing antibody effectively reduced BER activity to background levels for all periods tested (Fig. 5A, lanes 9-12). Next, this antibody was used to characterize the role of pol {lambda} in MEF extract-mediated BER. Cell extract from the pol {beta} -/- cell line was preincubated with preimmune serum (Fig. 5B, lanes 5-8) or antibody against pol {lambda} (lanes 9-12). As seen in Fig. 5B, preimmune serum had no significant impact on extract-mediated BER activity, whereas the neutralizing antibody against pol {lambda} reduced repair to a background level for all times examined.



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  		FIG. 6.
Demonstration of pol {lambda}-mediated back-up BER in MEF extracts. A, cell extracts from pol {lambda} +/+ (lanes 1-3) or pol {lambda} -/- (lanes 4-6) primary MEFs were incubated with preimmune serum (lanes 2 and 5) or with a neutralizing antibody against pol {beta} (lanes 3 and 6) for 30 min on ice and then an additional 10 min at 25 °C. After the preincubation, uracil-containing 35-base-pair duplex DNA was added, and repair reactions were performed for 10 min at 37 °C as described under "Experimental Procedures." Reaction products were analyzed as in Fig. 1. The position of the ligated BER product (35-mer) and unligated one-nucleotide (1-nt) insertion product are indicated. B, ligated BER products in lanes 3 and 6 were scanned by a PhosphorImager and quantified by using ImageQuant software. Products (PhosphorImager units) were plotted for each specific cell extract.

 
In the next experiments, we used extracts from pol {lambda} +/+ and pol {lambda} -/- cells incubated in the presence of pol {beta} antibody to evaluate the role of pol {lambda}. When the pol {lambda} +/+ cell extract was incubated with pol {beta} antibody a substantial level of BER persisted (Fig. 6A, lane 3). With the pol {lambda} -/- cell extract, the level of BER was much lower (Fig. 6A, lane 6) than that observed with the pol {lambda} +/+ cell extract (Fig. 6, A, lane 3, and B). We conclude from these results that pol {lambda} participates in MEF extract-mediated BER.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In planning this study, we reasoned that the biological significance of pol {lambda} in mammalian cell BER was an open question. An earlier study had demonstrated the capacity of the purified human pol {lambda} to function in an in vitro BER system with several other purified BER enzymes (17). This information, however, did not establish whether pol {lambda} could fulfill such a BER role in any particular cell type, such as mouse fibroblasts. The capacity to provide such a BER function depends on whether or not the enzyme is sufficiently expressed in these cells. This is an important issue in considering a role for pol {lambda} in BER because of its weak DNA polymerase activity (turnover number >30-fold lower than that of pol {beta}; data not shown). Even in cases in which pol {lambda} is expressed at a high level, the enzyme may not be active because of post-translational modifications or subcellular compartmentalization. Thus, for pol {lambda} to be active in the cell extracts studied here, the enzyme must be expressed at a substantial level, and it must be soluble, active, and available for recruitment to the BER substrate. None of these factors were addressed by the previous experiments with purified pol {lambda}. Extract-based biochemical and genetic experiments are needed to answer questions about the biological significance of pol {lambda}. In light of this, the results reported here significantly extend our biological understanding of pol {lambda}.

In the past, the importance of each BER-related enzyme in protection against DNA base damage has been studied using cytotoxicity assays with cells carrying a null mutation in the corresponding gene. In MEFs, a role of pol {beta} in protection against DNA-alkylating agents was exposed in this way (2, 30). Despite the pol {beta}-null cell hypersensitivity to DNA-alkylating agents, a significant level of resistance remains, pointing to the idea that pol {beta}-independent BER may exist in these cells. It was then found that replicative polymerases can also fulfill the polymerase role in BER (36-39), providing a back-up system for the pol {beta} deficiency. In addition, other mammalian DNA polymerases could play a role as a "back-up polymerase" in BER and, in particular, the pol {beta} relative pol {lambda} would seem to be an excellent candidate. Because pol {lambda} is related to pol {beta} and possesses two key biochemical activities that provide for a possible role in BER (dRP lyase and gap-filling synthesis) (16, 17), we ascertained whether pol {lambda} is capable of mediating a back-up BER activity in MEF extracts. This was pursued by examining the in vitro BER capacity of extracts prepared from pol {beta}-null MEFs. Indeed, when in vitro BER assays were performed using an extended incubation period (120 min), BER activity was detected (Fig. 1B, lanes 5-8). As has been well established, pol {beta} -/- cell extract possesses BER activity (4, 8, 39, 40). By making use of MEFs containing targeted deletions in either the pol {lambda} or pol {beta} gene and neutralizing antibodies against these proteins to suppress their activity, we showed that pol {lambda} -/- MEF extract had reduced BER capacity compared with the pol {lambda} +/+ MEF extract and that a neutralizing antibody against pol {lambda} can reduce pol {beta}-independent repair to background levels. We therefore conclude that pol {lambda} was capable of contributing to a portion of this back-up in vitro BER activity in MEF extracts.

With this new information on the capacity of pol {lambda} to function in BER, it should be interesting to examine cellular sensitivity to various DNA damaging agents to determine whether pol {lambda} plays a prominent role in the protection against different DNA lesions. This screening would be most effectively accomplished by the use of a pol {beta} -/-, pol {lambda} -/- double knock-out cell line. Double knock-out cell lines developed by either small interfering RNA technology or breeding of appropriate knock-out mouse lines may provide more insight into the role played by each individual enzyme in protection against different types of DNA damage.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: 111 T.W. Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-3267; Fax: 919-541-3592; E-mail: wilson5{at}niehs.nih.gov.

1 The abbreviations used are: BER, base excision repair; APE, apurinic/apyrimidinic endonuclease; dRP, 5'-deoxyribose phosphate; pol, polymerase; UDG, uracil-DNA glycosylase; Me2SO, dimethyl sulfoxide; MEF, mouse embryonic fibroblast; MOPS, 3-(N-morpholino)propanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Barbara Bertocci and Claude-Agnès Reynaud (Institut National de la Santé et de la Recherche Médicale, Paris, France) for the pol {lambda} +/+ and -/- primary mouse embryonic fibroblast cells; Drs. Miguel García-Díaz, Katarzyna Bebenek, and Thomas A. Kunkel (National Institute of Environmental Health Sciences) for valuable discussion; Drs. Miguel García-Díaz and Luis Blanco (Centro de Biología Molecular Severo Ochoa) for the neutralizing antibody against pol {lambda}; and Drs. Kenjiro Asagoshi, Katarzyna Bebenek, Miguel García-Díaz, Julie Horton, Scott McCulloch, and Yuan Liu for critical review of this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Krokan, H. E., Nilsen, H., Skorpen, F., Otterlei, M., and Slupphaug, G. (2000) FEBS Lett. 476, 73-77[CrossRef][Medline] [Order article via Infotrieve]
  2. Sobol, R. W., Horton, J. K., Kuhn, R., Gu, H., Singhal, R. K., Prasad, R., Rajewsky, K., and Wilson, S. H. (1996) Nature 379, 183-186[CrossRef][Medline] [Order article via Infotrieve]
  3. Horton, J. K., Joyce-Gray, D. F., Pachkowski, B. F., Swenberg, J. A., and Wilson, S. H. (2003) DNA Repair 2, 27-48[CrossRef][Medline] [Order article via Infotrieve]
  4. Fortini, P., Parlanti, E., Sidorkina, O. M., Laval, J., and Dogliotti, E. (1999) J. Biol. Chem. 274, 15230-15236[Abstract/Free Full Text]
  5. Nealon, K., Nicholl, I. D., and Kenny, M. K. (1996) Nucleic Acids Res. 24, 3763-3770[Abstract/Free Full Text]
  6. Singhal, R. K., Prasad, R., and Wilson, S. H. (1995) J. Biol. Chem. 270, 949-957[Abstract/Free Full Text]
  7. Dianov, G., Price, A., and Lindahl, T. (1992) Mol. Cell. Biol. 12, 1605-1612[Abstract/Free Full Text]
  8. Dianov, G., Bischoff, C., Piotrowski, J., and Bohr, V. A. (1998) J. Biol. Chem. 273, 33811-33816[Abstract/Free Full Text]
  9. Miller, M. R., and Chinault, D. N. (1982) J. Biol. Chem. 257, 10204-10209[Abstract/Free Full Text]
  10. Garcia-Diaz, M., Dominguez, O., Lopez-Fernandez, L. A., de Lera, L. T., Saniger, M. L., Ruiz, J. F., Parraga, M., Garcia-Ortiz, M. J., Kirchhoff, T., del Mazo, J., Bernad, A., and Blanco, L. (2000) J. Mol. Biol. 301, 851-867[CrossRef][Medline] [Order article via Infotrieve]
  11. Nagasawa, K., Kitamura, K., Yasui, A., Nimura, Y., Ikeda, K., Hirai, M., Matsukage, A., and Nakanishi, M. (2000) J. Biol. Chem. 275, 31233-31238[Abstract/Free Full Text]
  12. Prasad, R., Widen, S. G., Singhal, R. K., Watkins, J., Prakash, L., and Wilson, S. H. (1993) Nucleic Acids Res. 21, 5301-5307[Abstract/Free Full Text]
  13. Shimizu, K., Santocanale, C., Ropp, P. A., Longhese, M. P., Plevani, P., Lucchini, G., and Sugino, A. (1993) J. Biol. Chem. 268, 27148-27153[Abstract/Free Full Text]
  14. Aoufouchi, S., Flatter, E., Dahan, A., Faili, A., Bertocci, B., Storck, S., Delbos, F., Cocea, L., Gupta, N., Weill, J. C., and Reynaud, C. A. (2000) Nucleic Acids Res. 28, 3684-3693[Abstract/Free Full Text]
  15. Garcia-Diaz, M., Bebenek, K., Krahn, J. M., Blanco, L., Kunkel, T. A., and Pedersen, L. C. (2004) Mol. Cell 13, 561-572[CrossRef][Medline] [Order article via Infotrieve]
  16. Garcia-Diaz, M., Bebenek, K., Sabariegos, R., Dominguez, O., Rodriguez, J., Kirchhoff, T., Garcia-Palomero, E., Picher, A. J., Juarez, R., Ruiz, J. F., Kunkel, T. A., and Blanco, L. (2002) J. Biol. Chem. 277, 13184-13191[Abstract/Free Full Text]
  17. Garcia-Diaz, M., Bebenek, K., Kunkel, T. A., and Blanco, L. (2001) J. Biol. Chem. 276, 34659-34663[Abstract/Free Full Text]
  18. Lee, J. W., Blanco, L., Zhou, T., Garcia-Diaz, M., Bebenek, K., Kunkel, T. A., Wang, Z., and Povirk, L. F. (2004) J. Biol. Chem. 279, 805-811[Abstract/Free Full Text]
  19. Tseng, H. M., and Tomkinson, A. E. (2002) J. Biol. Chem. 277, 45630-45637[Abstract/Free Full Text]
  20. Wilson, T. E., and Lieber, M. R. (1999) J. Biol. Chem. 274, 23599-23609[Abstract/Free Full Text]
  21. Ramadan, K., Shevelev, I. V., Maga, G., and Hubscher, U. (2002) J. Biol. Chem. 277, 18454-18458[Abstract/Free Full Text]
  22. Ramadan, K., Maga, G., Shevelev, I. V., Villani, G., Blanco, L., and Hubscher, U. (2003) J. Mol. Biol. 328, 63-72[CrossRef][Medline] [Order article via Infotrieve]
  23. Strauss, P. R., Beard, W. A., Patterson, T. A., and Wilson, S. H. (1997) J. Biol. Chem. 272, 1302-1307[Abstract/Free Full Text]
  24. Abbotts, J., SenGupta, D. N., Zmudzka, B., Widen, S. G., Notario, V., and Wilson, S. H. (1988) Biochemistry 27, 901-909[CrossRef][Medline] [Order article via Infotrieve]
  25. Kumar, A., Widen, S. G., Williams, K. R., Kedar, P., Karpel, R. L., and Wilson, S. H. (1990) J. Biol. Chem. 265, 2124-2131[Abstract/Free Full Text]
  26. Slupphaug, G., Eftedal, I., Kavli, B., Bharati, S., Helle, N. M., Haug, T., Levine, D. W., and Krokan, H. E. (1995) Biochemistry 34, 128-138[CrossRef][Medline] [Order article via Infotrieve]
  27. Wang, Y. C., Burkhart, W. A., Mackey, Z. B., Moyer, M. B., Ramos, W., Husain, I., Chen, J., Besterman, J. M., and Tomkinson, A. E. (1994) J. Biol. Chem. 269, 31923-31928[Abstract/Free Full Text]
  28. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  29. Bertocci, B., De Smet, A., Flatter, E., Dahan, A., Bories, J. C., Landreau, C., Weill, J. C., and Reynaud, C. A. (2002) J. Immunol. 168, 3702-3706[Abstract/Free Full Text]
  30. Sobol, R. W., Prasad, R., Evenski, A., Baker, A., Yang, X. P., Horton, J. K., and Wilson, S. H. (2000) Nature 405, 807-810[CrossRef][Medline] [Order article via Infotrieve]
  31. Kedar, P. S., Kim, S. J., Robertson, A., Hou, E., Prasad, R., Horton, J. K., and Wilson, S. H. (2002) J. Biol. Chem. 277, 31115-31123[Abstract/Free Full Text]
  32. Horton, J. K., Baker, A., Berg, B. J., Sobol, R. W., and Wilson, S. H. (2002) DNA Repair 1, 317-333[Medline] [Order article via Infotrieve]
  33. Srivastava, D. K., Berg, B. J., Prasad, R., Molina, J. T., Beard, W. A., Tomkinson, A. E., and Wilson, S. H. (1998) J. Biol. Chem. 273, 21203-21209[Abstract/Free Full Text]
  34. Lee, M. Y., and Toomey, N. L. (1986) Nucleic Acids Res. 14, 1719-1726[Abstract/Free Full Text]
  35. Ling, H., Sayer, J. M., Plosky, B. S., Yagi, H., Boudsocq, F., Woodgate, R., Jerina, D. M., and Yang, W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2265-2269[Abstract/Free Full Text]
  36. Matsumoto, Y., Kim, K., and Bogenhagen, D. F. (1994) Mol. Cell. Biol. 14, 6187-6197[Abstract/Free Full Text]
  37. Biade, S., Sobol, R. W., Wilson, S. H., and Matsumoto, Y. (1998) J. Biol. Chem. 273, 898-902[Abstract/Free Full Text]
  38. Stucki, M., Pascucci, B., Parlanti, E., Fortini, P., Wilson, S. H., Hubscher, U., and Dogliotti, E. (1998) Oncogene 17, 835-843[CrossRef][Medline] [Order article via Infotrieve]
  39. Fortini, P., Pascucci, B., Parlanti, E., Sobol, R. W., Wilson, S. H., and Dogliotti, E. (1998) Biochemistry 37, 3575-3580[CrossRef][Medline] [Order article via Infotrieve]
  40. Fortini, P., Pascucci, B., Belisario, F., and Dogliotti, E. (2000) Nucleic Acids Res. 28, 3040-3046[Abstract/Free Full Text]

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